Compositions for bacterial mediated gene silencing and methods of using same

ABSTRACT

Methods are described for the delivery of one or more small interfering RNAs (siRNAs) to a eukaryotic cell using a bacterium. Methods are also described for using this bacterium to regulate gene expression in eukaryotic cells using RNA interference, and methods for treating cancer of cell proliferative disorders. The bacterium includes one or more siRNAs or one or more DNA molecules encoding one or more siRNAs. Vectors are also described for use with the bacteria of the invention for causing RNA interference in eukaryotic cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is continuation application of U.S. Ser. No.11/793,429, filed Nov. 20, 2007, which is a 35 U.S.C. §371 NationalPhase Application of PCT/US2005/045513, filed Dec. 16, 2005; whichclaims the benefit of, and priority to, U.S. Ser. No. 60/637,277 filedDec. 17, 2004 and U.S. Ser. No. 60/651,238 filed Feb. 8, 2005, each ofwhich is incorporated by reference in its entirety.

BACKGROUND

Gene silencing through RNAi (RNA-interference) by use of shortinterfering RNA (siRNA) has emerged as a powerful tool for molecularbiology and holds the potential to be used for therapeutic genesilencing. Short hairpin RNA (shRNA) transcribed from small DNA plasmidswithin the target cell has also been shown to mediate stable genesilencing and achieve gene knockdown at levels comparable to thoseobtained by transfection with chemically synthesized siRNA (T. R.Brummelkamp, R. Bernards, R. Agami, Science 296, 550 (2002), P. J.Paddison, A. A. Caudiy, G. J. Hannon, PNAS 99, 1443 (2002)).

Possible applications of RNAi for therapeutic purposes are extensive andinclude silencing and knockdown of disease genes such as oncogenes orviral genes. One major obstacle for the therapeutic use of RNAi is thedelivery of siRNA to the target cell (Zamore P D, Aronin N. NatureMedicine 9, (3):266-8 (2003)). In fact, delivery has been described asthe major hurdle now for RNAi (Phillip Sharp, cited by Nature newsfeature, Vol 425, 2003, 10-12)

Two methods have been described which can be used in mouse models:

(1) Direct hydrodynamic intravenous injection of siRNA or shRNA-encodingplasmids: using this method, several authors have described applicationof RNAi against various conditions, e.g. hepatitis B (A. P. McCaffrey etal., Nat Biotechnol. 2003 June; 21(6):639-44), fulminant hepatitis (E.Song, S. K. Lee, J. Wang, N. Ince, J. MM, J. Chen, P. Shankar, J.Lieberman. Nature Medicine 9, 347 (2003)), tumor xenograft (Spaenkuch B,et al. JNCI, 96(1): 862-72 (2004)), hepatic transgene expression (D. L.Lewis, J. E. Hagstrom, A. G. Loomis, J. A. Wolff, H. Hereijer, NatureGenetics, 32, 107 (2002), D. R. Sorensen D R, M. Leirdal, M. Sioud, JMB,327, 761 (2003)). This method uses a high pressure and high volumeinjection (2.5 ml) into the mouse tail vein. The mechanism of siRNA/DNAuptake into the cells is not clear but probably mechanical damage to thevascular endothelial layer is involved. A clear disadvantage of thismethod is that this is not a method which could be developed into humanapplication as it involves a massive volume charge and completelyunknown mechanism of action.

(2) Direct injection into the target tissue (brain) of an siRNA encodingadenoviral vector (H. Xia, Q. Mao, H. L. Paulson, B. L. Davidson, NatBiotechnol, 20, 1006 (2002)). This method showed silencing of transgene(GFP) expression in the brain tissues reached by the adenoviral vector.However, the area of silencing could not be predicted reliably. Thismethod might be developed further and might become applicable for local,e.g. intratumoral injection. Viral vectors have been used widely forgene therapy purposes, but one lesson learned from gene therapyexperiments is that viral spreading can be unpredictable at times andlead to unwanted side effects (Marshall E. Science 286(5448): 2244-5(1999)). A new method is needed for the safe and predictableadministration of interfering RNAs to mammals.

SUMMARY OF THE INVENTION

The invention generally pertains to methods of delivering one or moresiRNAs to a eukaryotic cell by introducing a bacterium to the cell,wherein the bacterium contains one or more siRNAs or one or more DNAmolecules encoding one or more siRNAs.

In one embodiment of this method, the eukaryotic cell is in vivo. Inanother embodiment of this invention, the eukaryotic cell is in vitro.

The invention also pertains to a method of regulating gene expression ina eukaryotic cell, by introducing a bacterium to the cell, wherein thebacterium contains one or more siRNAs or one or more DNA moleculesencoding one or more siRNAs, wherein the expressed siRNAs interfere withthe mRNA of the gene to be regulated, thereby regulating expression ofthe gene.

In one embodiment of this method, the expressed siRNAs direct themultienzyme complex RISC (RNA-induced silencing complex) of the cell tointeract with the mRNA to be regulated. This complex degrades the mRNA.This causes the expression of the gene to be decreased or inhibited. Inanother embodiment of this method, the gene is ras or β-catenin. In oneaspect of this embodiment, the ras is k-Ras.

In one embodiment of the above methods of the invention, the eukaryoticcell is a mammalian cell. In one aspect of this embodiment, themammalian cell is a human cell.

The invention also pertains to a method of treating or preventing canceror a cell proliferation disorder in a mammal, by regulating theexpression of a gene or several genes in a cell known to increase cellproliferation by introducing a bacterium to the cell. The bacteriumcontains one or more siRNAs or one or more DNA molecules encoding one ormore siRNAs.

In one embodiment of this method of the invention, the mammal is ahuman. In another embodiment of this method the expressed siRNAsinterfere with the mRNA of the gene to be regulated. In one aspect ofthis embodiment, the expressed siRNAs direct the multienzyme complexRISC (RNA-induced silencing complex) of the cell to interact with themRNA to be regulated. This complex degrades the mRNA. This causes theexpression of the gene to be decreased or inhibited.

In another embodiment of this method, the gene is ras or β-catenin. Inone aspect of this embodiment, the ras is k-Ras.

In another embodiment of this method of the invention, the cell is acolon cancer cell or a pancreatic cancer cell. In one aspect of thisembodiment, the colon cancer cell is an SW 480 cell. In another aspectof this embodiment, the pancreatic cancer cell is a CAPAN-1 cell.

In one embodiment of the above methods of the invention, the bacteriumis non-pathogenic or non-virulent. In another aspect of this embodiment,the bacterium is therapeutic. In another aspect of this embodiment, thebacterium is an attenuated strain selected from the group consisting ofListeria, Shigella, Salmonella, E. coli, and Bifidobacteriae.Optionally, the Salmonella strain is an attenuated strain of theSalmonella typhimurium species. Optionally, the Salmonella typhimuriumstrain is SL 7207 or VNP20009. Optionally, the E. coli strain is BM2710.

In another embodiment of the above methods of the invention, the one ormore DNA molecules encoding the one or more siRNAs are transcribedwithin the eukaryotic cell. In one aspect of this embodiment, the one ormore siRNAs are transcribed within the eukaryotic cells as shRNAs. Inanother aspect of this embodiment, the one or more DNA moleculesencoding the one or more siRNAs contains an RNA-polymerase III promoter.Optionally, the RNA polymerase III promoter is a U6 promoter or an H1promoter.

In another embodiment of the above methods of the invention, the one ormore DNA molecules encoding one or more siRNAs are transcribed withinthe bacterium. In one aspect of this embodiment, the one or more DNAmolecules contain a prokaryotic promoter. Optionally, the prokaryoticpromoter is a T7 promoter.

In another embodiment of the above methods of the invention, the one ormore DNA molecules are introduced to the eukaryotic cell through typeIII export or bacterial lysis. In one aspect of this embodiment, thebacterial lysis is triggered by the addition of an intracellular activeantibiotic. Optionally, the antibiotic is tetracycline. In anotheraspect of this embodiment, the bacterial lysis is triggered throughbacterial metabolic attenuation. Optionally, the metabolic attenuationis auxotrophy.

The invention also pertains to a bacterium containing one or more siRNAsor one or more DNA molecules encoding one or more siRNAs.

In one embodiment of this invention, the bacterium is a non-pathogenicor a non-virulent bacterium. In another aspect of this embodiment, thebacterium is a therapeutic bacterium.

In another embodiment of this invention, the bacterium is an attenuatedstrain selected from a member of the group consisting of Listeria,Shigella, Salmonella, E. coli, and Bifidobacteriae. Optionally, theSalmonella strain is an attenuated strain of the Salmonella typhimuriumspecies. Optionally, the Salmonella typhimurium strain is SL 7207 orVNP20009. Optionally, the E. coli strain is BM 2710.

The invention also pertains to a prokaryotic vector containing a DNAencoding one or more siRNAs and an RNA-polymerase III compatiblepromoter or a prokaryotic promoter.

In one embodiment of this vector of the invention, the RNA polymeraseIII promoter is a U6 promoter or an H1 promoter. In another embodimentof this vector of the invention, the prokaryotic promoter is a T7promoter.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows micrographs of invasion of SL 7207 into SW 480 cells.

FIG. 1B shows FACS analysis of a knockdown of green fluorescent proteinexpression in CRL 2583 cells.

FIG. 1C shows micrographs showing loss of fluorescence in CRL 2583cells.

FIG. 2A shows Western blots of k-Ras and β-catenin in SW480 cells.

FIG. 2B is a series of bar charts showing viability of SW480 cells underSL-siRAS treatment (top) and combined treatment SL-siCAT and SL-siRAS(bottom).

FIG. 2C is a series of bar charts showing viability of SW480 cells underSL-siCAT treatment (top) and colony formation efficiency % (bottom).

FIG. 2D shows photographs of tumorigenicity in nude mice injected withSW480 cells transfected with various siRNAs.

FIG. 3A shows micrographs of transgenic mouse liver sections.

FIG. 3B shows flow cytometry measurements of hepatocyte and splenocytesuspensions.

FIG. 4A shows Western blots of k-Ras and β-catenin from SW480 cellstransfected with silencer plasmid. FIG. 4B shows Western blots ofβ-catenin.

FIG. 5 shows micrographs of histochemical staining of liver sections ofmice showing changes in GFP expression levels.

FIG. 6A is a schematic showing the Transkingdom RNA Interference Plasmid(TRIP).

FIG. 6B is a photograph showing an immunoblot of β-catenin from SW480cells transfected with TRIP.

FIG. 6C is a photograph showing an immunoblot of β-catenin from SW480cells transfected with TRIP for exposure time from 30 to 120 minutes.

FIG. 6D shows an RT-PCR photograph of β-catenin and k-Ras mRNA fromSW480 cells transfected with TRIP.

FIG. 6E is a photograph showing an immunoblot of k-Ras in SW480 and DLD1cells following transfection with a TRIP against mutant k-Ras (GGT-->GTTat codon 12) mutant k-Ras (GGC-->GAC at codon 13).

FIG. 6F is a photograph showing an immunoblot of SW480 cells transfectedwith a TRIP against wild type k-Ras.

FIG. 7A is a photograph of RT-PCR showing β-catenin silencing followingtreatment with E. coli expressed shRNAs.

FIG. 7B is schematic showing specific cleavage sites in β-catenin, andshowing SEQ ID NO:16.

FIG. 7C is a photograph of a 5′-RACE-PCR showing specific cleavageproducts.

FIG. 7D is a photograph of a blot showing the mRNA expression of variousgenes.

FIG. 8A is a photograph of cellular staining showing that both Inv andHly are required for bacterial entry.

FIG. 8B is a photograph of an RNA blot showing that TRIP lacking Hly isunable to induce knockdown of a target gene.

FIG. 8C is a photograph of an RNA blot showing that both Inv and Hly arerequired to facilitate efficient transkingdom iRNA.

FIG. 8D is a photograph of an RNA blot showing the effect of delayedaddition of tetracycline on gene silencing.

FIG. 8E is a photograph of cellular staining showing lack of significantbacterial replication in the absence of antibiotics beyond 2 hincubation.

FIG. 9A is a graph showing that oral administration of E. coliexpressing shRNA against β-catenin in mice leads to significantreduction of β-catenin expression in the intestinal epithelium. FIG. 9Bis a photograph of immunohistochemistry staining of intestinalepithelium with or without treatment. FIG. 9C is a graph showing adecrease in β-catenin mRNA expression following treatment. FIG. 9D is agraph showing a decrease in β-catenin protein expression followingtreatment. FIG. 9E is a photograph of immunohistochemistry stainingshowing decrease in β-catenin protein expression following treatment.

FIG. 10 is a photograph of immunohistochemistry staining showing thatGAPDH expression is not altered by E. coli expressing shRNA againstβ-catenin after oral dosing in mice.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains to methods of delivering small interfering RNAs(siRNAs) to eukaryotic cells using non-pathogenic or therapeutic strainsof bacteria. The bacteria deliver RNA encoding DNA or RNA, itself, toeffect RNA interference (RNAi). The interfering RNA of the inventionregulates gene expression in eukaryotic cells. It silences or knocksdown genes of interest inside target cells. The interfering RNA directsthe cell-owned multienzyme-complex RISC (RNA-induced silencing complex)to the mRNA of the gene to be silenced. Interaction of RISC and mRNAresults in degradation of the mRNA. This leads to effectivepost-transcriptional silencing of the gene of interest. This method isreferred to as Bacteria Mediated Gene Silencing (BMGS).

Bacterial delivery is more attractive than viral delivery as it can becontrolled by use of antibiotics and attenuated bacterial strains whichare unable to multiply. Also, bacteria are much more accessible togenetic manipulation which allows the production of vector strainsspecifically tailored to certain applications. In one embodiment of theinvention, the methods of the invention are used to create bacteriawhich cause RNAi in a tissue specific manner.

The siRNA is either introduced into the target cell directly or bytransfection or can be transcribed within the target cell ashairpin-structured dsRNA (shRNA) from specific plasmids withRNA-polymerase III compatible promoters (U6, H1) (P. J. Paddison, A. A.Caudiy, G. J. Hannon, PNAS 99, 1443 (2002), T. R. Brummelkamp, R.Bernards, R. Agami, Science 296, 550 (2002)).

Liberation of siRNA encoding plasmid from the intracellular bacteriaoccurs through active mechanisms. One mechanism involves the type IIIexport system in S. typhimuriumm, a specialised multiprotein complexspanning the bacterial cell membrane whose functions include secretionof virulence factors to the outside of the cell to allow signalingtowards the target cell, but which can also be used to deliver antigensinto target cells. (Rüssmann H. Int J Med Microbiol, 293:107-12 (2003))or through bacterial lysis and liberation of bacterial contents into thecytoplasm. The lysis of intracellular bacteria is triggered throughaddition of an intracellularly active antibiotic (tetracycline) oroccurs naturally through bacterial metabolic attenuation (auxotrophy).After liberation of the eukaryotic transcription plasmid, shRNA or siRNAare produced within the target cell and trigger the highly specificprocess of mRNA degradation, which results in silencing of the targetedgene.

The non-virulent bacteria of the invention have invasive properties andmay enter a mammalian host cell through various mechanisms. In contrastto uptake of bacteria by professional phagocytes, which normally resultsin the destruction of the bacterium within a specialized lysosome,invasive bacteria strains have the ability to invade non-phagocytic hostcells. Naturally occurring examples of such bacteria are intracellularpathogens such as Listeria, Shigella and Salmonella, but this propertycan also be transferred to other bacteria such as E. coli andBifidobacteriae, including probiotics through transfer ofinvasion-related genes (P. Courvalin, S. Goussard, C. Grillot-Courvalin,C.R.Acad.Sci.Paris 318, 1207 (1995)). In other embodiments of theinvention, bacteria used to deliver interfering RNAs to host cellsinclude Shigella flexneri (D. R. Sizemore, A. A. Branstrom, J. C.Sadoff, Science 270, 299 (1995)), invasive E. coli (P. Courvalin, S.Goussard, C. Grillot-Courvalin, C.R.Acad.Sci.Paris 318, 1207 (1995), C.Grillot-Courvalin, S. Goussard, F. Huetz, D. M. Ojcius, P. Courvalin,Nat Biotechnol 16, 862 (1998)), Yersinia enterocolitica (A. Al-Mariri A,A. Tibor, P. Lestrate, P. Mertens, X. De Bolle, J. J. Letesson InfectImmun 70, 1915 (2002)) and Listeria monocytogenes (M. Hense, E. Domann,S. Krusch, P. Wachholz, K. E. Dittmar, M. Rohde, J. Wehland, T.Chakraborty, S. Weiss, Cell Microbiol 3, 599 (2001), S. Pilgrim, J.Stritzker, C. Schoen, A. Kolb-Maurer, G. Geginat, M. J. Loessner, I.Gentschev, W. Goebel, Gene Therapy 10, 2036 (2003)). Any invasivebacterium is useful for DNA transfer into eukaryotic cells (S. Weiss, T.Chakraborty, Curr Opinion Biotechnol 12, 467 (2001)).

BMGS is performed using the naturally invasive pathogen Salmonellatyphimurium. In one aspect of this embodiment, the strains of Salmonellatyphimurium include SL 7207 and VNP20009 (S. K. Hoiseth, B. A. D.Stocker, Nature 291, 238 (1981); Pawelek J M, Low K B, Bermudes D.Cancer Res. 57(20):4537-44 (Oct. 15, 1997)). In another embodiment ofthe invention, BMGS is performed using attenuated E. coli. In one aspectof this embodiment, the strain of E. coli is BM 2710 (C.Grillot-Courvalin, S. Goussard, F. Huetz, D. M. Ojcius, P. Courvalin,Nat Biotechnol 16, 862 (1998)). In another aspect of this embodiment,the BM 2710 strain is engineered to possess cell-invading propertiesthrough an invasion plasmid. In one aspect of the invention, thisplasmid is pGB2inv-hly.

A double “trojan horse” technique is also used with an invasive andauxotrophic bacterium carrying a eukaryotic transcription plasmid. Thisplasmid is, in turn, transcribed by the target cell to form a hairpinRNA structure that triggers the intracellular process of RNAi. Thismethod of the invention induces significant gene silencing of a varietyof genes. In certain aspects of this embodiment, the genes include atransgene (GFP), a mutated oncogene (k-Ras) and a cancer related gene(β-catenin) in vitro.

The invention also pertains to a variation of the described method,termed Bacteria Transcribed Gene Silencing (BTGS). In this aspect of theinvention, siRNA is directly produced by the invasive bacteria asopposed to the target cell. A transcription plasmid controlled by aprokaryotic promoter (e.g. T7) is inserted into the carrier bacteriathrough standard transformation protocols. siRNA is produced within thebacteria and is liberated within the mammalian target cell afterbacterial lysis triggered either by auxotrophy or by timed addition ofantibiotics.

The RNAi methods of the invention, including BMGS and BTGS are used as acancer therapy or to prevent cancer. This method is effected bysilencing or knocking down genes involved with cell proliferation orother cancer phenotypes. Examples of these genes are k-Ras andβ-catenin. Specifically, k-Ras and β-catenin are targets for RNAi basedtherapy of colon cancer. These oncogenes are active and relevant in themajority of clinical cases. BMGS is applied to reach the intestinaltract for colon cancer treatment and prevention. These methods are alsoused to treat of animals carrying xenograft tumors, to treat and preventcancer in k-Ras V12 model of intestinal tumorgenesis, and to prevent andtreat tumors in the adenomatous polyposis coli min mouse model (APC-minmodel) In this model, the mouse has a defective APC gene resulting inthe formation of numerous intestinal and colonic polyps which is used asan animal model for human familiar adenomatous polyposis coli (FAP) ofintestinal tumorigenesis.

The invention also encompasses a prokaryotic shRNA-encodingtranscription plasmid for use with invasive bacteria to performBacteria-Transcribed Gene Silencing (BTGS). These plasmids are used toscreen different cancer-related targets in transgenic as well as wildtype animals for therapeutic experiments.

The RNAi methods of the invention, including BMGS and BTGS are also usedto treat or prevent viral diseases (e.g. hepatitis) and geneticdisorders.

The RNAi methods of the invention, including BMGS and BTGS are also usedto create cancer-preventing “probiotic bacteria” for use, especiallywith the target of GI tract or liver.

The RNAi methods of the invention, including BMGS and BTGS are used astherapy against inflammatory conditions, e.g. hepatitis, inflammatorybowel disease (IBD) or colitis. These methods are used to silence orknockdown non-cancer gene targets (viral genes, for treatment andprevention of hepatitis B, C; inflammatory genes, for treatment andprevention of inflammatory bowel disease) and others.

The RNAi methods of the invention, including BMGS and BTGS are used tocreate transient “knockdown” genetic animal models as opposed togenetically engineered knockout models to discover gene functions. Themethods are also used as in vitro transfection tool for research anddrug development

These methods use bacteria with desirable properties (invasiveness,attenuation, steerability) for example, Bifidobacteria and Listeria, areused to perform BMGS and BTGS. Invasiveness as well as eukaryotic orprokaryotic transcription of one or several shRNA is conferred to abacterium using plasmids.

The RNAi methods of the invention, including BMGS and BTGS are used fordelivery of gene silencing to the gut and colon, and for oralapplication in the treatment of various diseases, namely colon cancertreatment and prevention. In another aspect of this embodiment, deliveryof gene silencing is extra-intestinal.

1. Bacteria Delivering RNA to Eukaryotic Cells

According to the invention, any microorganism which is capable ofdelivering a molecule, e.g., an RNA molecule, into the cytoplasm of atarget cell, such as by traversing the membrane and entering thecytoplasm of a cell, can be used to deliver RNA to such cells. In apreferred embodiment, the microorganism is a prokaryote. In an even morepreferred embodiment, the prokaryote is a bacterium. Also within thescope of the invention are microorganisms other than bacteria which canbe used for delivering RNA to a cell. For example, the microorganism canbe a fungus, e.g., Cryptococcus neoformans, protozoan, e.g., Trypanosomacruzi, Toxoplasma gondii, Leishmania donovani, and plasmodia.

As used herein, the term “invasive” when referring to a microorganism,e.g., a bacterium, refers to a microorganism which is capable ofdelivering at least one molecule, e.g., an RNA or RNA-encoding DNAmolecule, to a target cell. An invasive microorganism can be amicroorganism which is capable of traversing a cell membrane, therebyentering the cytoplasm of said cell, and delivering at least some of itscontent, e.g., RNA or RNA-encoding DNA, into the target cell. Theprocess of delivery of the at least one molecule into the target cellpreferably does not significantly modify the invasion apparatus.

In a preferred embodiment, the microorganism is a bacterium. A preferredinvasive bacterium is a bacterium which is capable of delivering atleast one molecule, e.g., an RNA or RNA-encoding DNA molecule, to atarget cells, such as by entering the cytoplasm of a eukaryotic cell.Preferred invasive bacteria are live bacteria, e.g., live invasivebacteria.

Invasive microorganisms include microorganisms that are naturallycapable of delivering at least one molecule to a target cell, such as bytraversing the cell membrane, e.g., a eukaryotic cell membrane, andentering the cytoplasm, as well as microorganisms which are notnaturally invasive and which have been modified, e.g., geneticallymodified, to be invasive. In another preferred embodiment, amicroorganism which is not naturally invasive can be modified to becomeinvasive by linking the bacterium to an “invasion factor”, also termed“entry factor” or “cytoplasm-targeting factor”. As used herein, an“invasion factor” is a factor, e.g., a protein or a group of proteinswhich, when expressed by a non-invasive bacterium, render the bacteriuminvasive. As used herein, an “invasion factor” is encoded by a“cytoplasm-targeting gene”.

Naturally invasive microorganisms, e.g., bacteria, may have a certaintropism, i.e., preferred target cells. Alternatively, microorganisms,e.g., bacteria can be modified, e.g., genetically, to mimic the tropismof a second microorganism.

Delivery of at least one molecule into a target cell can be determinedaccording to methods known in the art. For example, the presence of themolecule, by the decrease in expression of an RNA or protein silencedthereby, can be detected by hybridization or PCR methods, or byimmunological methods which may include the use of an antibody.

Determining whether a microorganism is sufficiently invasive for use inthe invention may include determining whether sufficient RNA, wasdelivered to host cells, relative to the number of microorganismscontacted with the host cells. If the amount of RNA, is low relative tothe number of microorganisms used, it may be desirable to further modifythe microorganism to increase its invasive potential.

Bacterial entry into cells can be measured by various methods.Intracellular bacteria survive treatment by aminoglycoside antibiotics,whereas extracellular bacteria are rapidly killed. A quantitativeestimate of bacterial uptake can be achieved by treating cell monolayerswith the antibiotic gentamicin to inactivate extracellular bacteria,then by removing said antibiotic before liberating the survivingintracellular organisms with gentle detergent and determining viablecounts on standard bacteriological medium. Furthermore, bacterial entryinto cells can be directly observed, e.g., by thin-section-transmissionelectron microscopy of cell layers or by immunofluorescent techniques(Falkow et al. (1992) Annual Rev. Cell Biol. 8:333). Thus, varioustechniques can be used to determine whether a specific bacteria iscapable of invading a specific type of cell or to confirm bacterialinvasion following modification of the bacteria, such modification ofthe tropism of the bacteria to mimic that of a second bacterium.

Bacteria that can be used for delivering RNA according to the method ofthe invention are preferably non-pathogenic. However, pathogenicbacteria can also be used, so long as their pathogenicity has beenattenuated, to thereby render the bacteria non-harmful to a subject towhich it is administered. As used herein, the term “attenuatedbacterium” refers to a bacterium that has been modified to significantlyreduce or eliminate its harmfulness to a subject. A pathogenic bacteriumcan be attenuated by various methods, set forth below.

Without wanting to be limited to a specific mechanism of action, thebacterium delivering the RNA into the eukaryotic cell can enter variouscompartments of the cell, depending on the type of bacterium. Forexample, the bacterium can be in a vesicle, e.g., a phagocytic vesicle.Once inside the cell, the bacterium can be destroyed or lysed and itscontents delivered to the eukaryotic cell. A bacterium can also beengineered to express a phagosome degrading enyzme to allow leakage ofRNA from the phagosome. In some embodiments, the bacterium can stayalive for various times in the eukaryotic cell and may continue toproduce RNA. The RNA or RNA-encoding DNA can then be released from thebacterium into the cell by, e.g., leakage. In certain embodiments of theinvention, the bacterium can also replicate in the eukaryotic cell. In apreferred embodiment, bacterial replication does not kill the host cell.The invention is not limited to delivery of RNA or RNA-encoding DNA by aspecific mechanism and is intended to encompass methods and compositionspermitting delivery of RNA or RNA-encoding DNA by a bacteriumindependently of the mechanism of delivery.

Set forth below are examples of bacteria which have been described inthe literature as being naturally invasive (section 1.1), as well asbacteria which have been described in the literature as being naturallynon-invasive bacteria (section 1.2), as well as bacteria which arenaturally non-pathogenic or which are attenuated. Although some bacteriahave been described as being non-invasive (section 1.2), these may stillbe sufficiently invasive for use according to the invention. Whethertraditionally described as naturally invasive or non-invasive, anybacterial strain can be modified to modulate, in particular to increase,its invasive characteristics (e.g., as described in section 1.3).

1.1 Naturally Invasive Bacteria

The particular naturally invasive bacteria employed in the presentinvention is not critical thereto. Examples of such naturally-occurringinvasive bacteria include, but are not limited to, Shigella spp.,Salmonella spp., Listeria spp., Rickettsia spp., and enteroinvasiveEscherichia coli.

The particular Shigella strain employed is not critical to the presentinvention. Examples of Shigella strains which can be employed in thepresent invention include Shigella flexneri 2a (ATCC No. 29903),Shigella sonnei (ATCC No. 29930), and Shigella disenteriae (ATCC No.13313). An attenuated Shigella strain, such as Shigella flexneri 2a2457T aroA virG mutant CVD 1203 (Noriega et al. supra), Shigellaflexneri M90T icsA mutant (Goldberg et al. Infect. Immun., 62:5664-5668(1994)), Shigella flexneri Y SFL114 aroD mutant (Karnell et al. Vacc.,10:167-174 (1992)), and Shigella flexneri aroA aroD mutant (Verma et al.Vacc., 9:6-9 (1991)) are preferably employed in the present invention.Alternatively, new attenuated Shigella spp. strains can be constructedby introducing an attenuating mutation either singularly or inconjunction with one or more additional attenuating mutations.

At least one advantage to Shigella RNA vaccine vectors is their tropismfor lymphoid tissue in the colonic mucosal surface. In addition, theprimary site of Shigella replication is believed to be within dendriticcells and macrophages, which are commonly found at the basal lateralsurface of M cells in mucosal lymphoid tissues (reviewed by McGhee, J.R. et al. (1994) Reproduction, Fertility, & Development 6:369; Pascual,D. W. et al. (1994) Immunomethods 5:56). As such, Shigella vectors mayprovide a means to express antigens in these professional antigenpresenting cells. Another advantage of Shigella vectors is thatattenuated Shigella strains deliver nucleic acid reporter genes in vitroand in vivo (Sizemore, D. R. et al. (1995) Science 270:299; Courvalin,P. et al. (1995) Comptes Rendus de l Academie des Sciences SerieIII-Sciences de la Vie-Life Sciences 318:1207; Powell, R. J. et al.(1996) In: Molecular approaches to the control of infectious diseases.F. Brown, E. Norrby, D. Burton and J. Mekalanos, eds. Cold Spring HarborLaboratory Press, New York. 183; Anderson, R. J. et al. (1997) Abstractsfor the 97th General Meeting of the American Society forMicrobiology:E.). On the practical side, the tightly restricted hostspecificity of Shigella stands to prevent the spread of Shigella vectorsinto the food chain via intermediate hosts. Furthermore, attenuatedstrains that are highly attenuated in rodents, primates and volunteershave been developed (Anderson et al. (1997) supra; Li, A. et al. (1992)Vaccine 10:395; Li, A. et al. (1993) Vaccine 11:180; Karnell, A. et al.(1995) Vaccine 13:88; Sansonetti, P. J. and J. Arondel (1989) Vaccine7:443; Fontaine, A. et al. (1990) Research in Microbiology 141:907;Sansonetti, P. J. et al. (1991) Vaccine 9:416; Noriega, F. R. et al.(1994) Infection & Immunity 62:5168; Noriega, F. R. et al. (1996)Infection & Immunity 64:3055; Noriega, F. R. et al. (1996) Infection &Immunity 64:23; Noriega, F. R. et al. (1996) Infection & Immunity64:3055; Kotloff, K. L. et al. (1996) Infection & Immunity 64:4542).This latter knowledge will allow the development of well toleratedShigella vectors for use in humans.

Attenuating mutations can be introduced into bacterial pathogens usingnon-specific mutagenesis either chemically, using agents such asN-methyl-N′-nitro-N-nitrosoguanidine, or using recombinant DNAtechniques; classic genetic techniques, such as Tn10 mutagenesis,P22-mediated transduction, λ, phage mediated crossover, andconjugational transfer; or site-directed mutagenesis using recombinantDNA techniques. Recombinant DNA techniques are preferable since strainsconstructed by recombinant DNA techniques are far more defined. Examplesof such attenuating mutations include, but are not limited to:

(i) auxotrophic mutations, such as aro (Hoiseth et al. Nature,291:238-239 (1981)), gua (McFarland et al. Microbiol. Path., 3:129-141(1987)), nad (Park et al. J. Bact., 170:3725-3730 (1988), thy (Nnalue etal. Infect. Immun., 55:955-962 (1987)), and asd (Curtiss, supra)mutations;

(ii) mutations that inactivate global regulatory functions, such as cya(Curtiss et al. Infect. Immun., 55:3035-3043 (1987)), crp (Curtiss et al(1987), supra), phoP/phoQ (Groisman et al. Proc. Natl. Acad. Sci., USA,86:7077-7081 (1989); and Miller et al. Proc. Natl. Acad. Sci., USA,86:5054-5058 (1989)), phop^(c) (Miller et al. J. Bact., 172:2485-2490(1990)) or ompR (Dorman et al. Infect. Immun., 57:2136-2140 (1989))mutations;

(iii) mutations that modify the stress response, such as recA (Buchmeieret al. Mol. Micro., 7:933-936 (1993)), htrA (Johnson et al. Mol. Micro.,5:401-407 (1991)), htpR (Neidhardt et al. Biochem. Biophys. Res. Com.,100:894-900 (1981)), hsp (Neidhardt et al. Ann. Rev. Genet., 18:295-329(1984)) and groEL (Buchmeier et al. Sci., 248:730-732 (1990)) mutations;

(iv) mutations in specific virulence factors, such as IsyA (Libby et al.Proc. Natl. Acad. Sci., USA, 91:489-493 (1994)), pag or prg (Miller etal (1990), supra; and Miller et al (1989), supra), iscA or virG(d'Hauteville et al. Mol. Micro., 6:833-841 (1992)), plcA (Mengaud etal. Mol. Microbiol., 5:367-72 (1991); Camilli et al. J. Exp. Med,173:751-754 (1991)), and act (Brundage et al. Proc. Natl. Acad. Sci.,USA, 90:11890-11894 (1993)) mutations;

(v) mutations that affect DNA topology, such as topA (Galan et al.Infect. Immun., 58:1879-1885 (1990));

(vi) mutations that disrupt or modify the cell cycle, such as min (deBoer et al. Cell, 56:641-649 (1989)).

(vii) introduction of a gene encoding a suicide system, such as sacB(Recorbet et al. App. Environ. Micro., 59:1361-1366 (1993); Quandt etal. Gene, 127:15-21 (1993)), nuc (Ahrenholtz et al. App. Environ.Micro., 60:3746-3751 (1994)), hok, gef, kil, or phlA (Molin et al. Ann.Rev. Microbiol., 47:139-166 (1993));

(viii) mutations that alter the biogenesis of lipopolysaccharide and/orlipid A, such as rFb (Raetz in Esherishia coli and Salmonellatyphimurium, Neidhardt et al., Ed., ASM Press, Washington D.C. pp1035-1063 (1996)), galE (Hone et al. J. Infect. Dis., 156:164-167(1987)) and htrB (Raetz, supra), msbB (Reatz, supra)

(ix) introduction of a bacteriophage lysis system, such as lysogensencoded by P22 (Rennell et al. Virol, 143:280-289 (1985)), λ, mureintransglycosylase (Bienkowska-Szewczyk et al. Mol. Gen. Genet.,184:111-114 (1981)) or S-gene (Reader et al. Virol, 43:623-628 (1971));and

The attenuating mutations can be either constitutively expressed orunder the control of inducible promoters, such as the temperaturesensitive heat shock family of promoters (Neidhardt et al. supra), orthe anaerobically induced nirB promoter (Harborne et al. Mol. Micro.,6:2805-2813 (1992)) or repressible promoters, such as uapA (Gorfinkielet al. J. Biol. Chem., 268:23376-23381 (1993)) or gcv (Stauffer et al.J. Bact., 176:6159-6164 (1994)).

The particular Listeria strain employed is not critical to the presentinvention. Examples of Listeria strains which can be employed in thepresent invention include Listeria monocytogenes (ATCC No. 15313).Attenuated Listeria strains, such as L. monocytogenes actA mutant(Brundage et al. supra) or L. monocytogenes plcA (Camilli et al. J. Exp.Med., 173:751-754 (1991)) are preferably used in the present invention.Alternatively, new attenuated Listeria strains can be constructed byintroducing one or more attenuating mutations in groups (i) to (vii) asdescribed for Shigella spp. above.

The particular Salmonella strain employed is not critical to the presentinvention. Examples of Salmonella strains which can be employed in thepresent invention include Salmonella typhi (ATCC No. 7251) and S.typhimurium (ATCC No. 13311). Attenuated Salmonella strains arepreferably used in the present invention and include S. typhi-aroC-aroD(Hone et al. Vacc. 9:810 (1991) and S. typhimurium-aroA mutant(Mastroeni et al. Micro. Pathol. 13:477 (1992)). Alternatively, newattenuated Salmonella strains can be constructed by introducing one ormore attenuating mutations as described fro Shigella spp. above.

The particular Rickettsia strain employed is not critical to the presentinvention. Examples of Rickettsia strains which can be employed in thepresent invention include Rickettsia Rickettsiae (ATCC Nos. VR149 andVR891), Ricketsia prowaseckii (ATCC No. VR233), Rickettsia tsutsugamuchi(ATCC Nos. VR312, VR150 and VR609), Rickettsia mooseri (ATCC No. VR144),Rickettsia sibirica (ATCC No. VR151), and Rochalimaea quitana (ATCC No.VR358). Attenuated Rickettsia strains are preferably used in the presentinvention and can be constructed by introducing one or more attenuatingmutations in groups (i) to (vii) as described for Shigella spp. above.

The particular enteroinvasive Escherichia strain employed is notcritical to the present invention. Examples of enteroinvasiveEscherichia strains which can be employed in the present inventioninclude Escherichia coli strains 4608-58, 1184-68, 53638-C-17, 13-80,and 6-81 (Sansonetti et al. Ann. Microbiol. (Inst. Pasteur),132A:351-355 (1982)). Attenuated enteroinvasive Escherichia strains arepreferably used in the present invention and can be constructed byintroducing one or more attenuating mutations in groups (i) to (vii) asdescribed for Shigella spp. above.

Furthermore, since certain microorganisms other than bacteria can alsointeract with integrin molecules (which are receptors for certaininvasion factors) for cellular uptake, such microorganisms can also beused for introducing RNA into target cells. For example, viruses, e.g.,foot-and-mouth disease virus, echovirus, and adenovirus, and eukaryoticpathogens, e.g., Histoplasma capsulatum and Leishmania major interactwith integrin molecules.

1.2 Less Invasive Bacteria

Examples of bacteria which can be used in the invention and which havebeen described in the literature as being non-invasive or at least lessinvasive than the bacteria listed in the previous section (1.1) include,but are not limited to, Yersinia spp., Escherichia spp., Klebsiellaspp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesellaspp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilusspp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcusspp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp.,and Erysipelothrix spp. It may be necessary to modify these bacteria toincrease their invasive potential.

The particular Yersinia strain employed is not critical to the presentinvention. Examples of Yersinia strains which can be employed in thepresent invention include Y. enterocolitica (ATCC No. 9610) or Y. pestis(ATCC No. 19428). Attenuated Yersinia strains, such as Y. enterocoliticaYe03-R2 (al-Hendy et al. Infect. Immun., 60:870-875 (1992)) or Y.enterocolitica aroA (O'Gaora et al. Micro. Path., 9:105-116 (1990)) arepreferably used in the present invention. Alternatively, new attenuatedYersinia strains can be constructed by introducing one or moreattenuating mutations in groups (i) to (vii) as described for Shigellaspp. above.

The particular Escherichia strain employed is not critical to thepresent invention. Examples of Escherichia strains which can be employedin the present invention include E. coli H10407 (Elinghorst et al.Infect. Immun., 60:2409-2417 (1992)), and E. coli EFC4, CFT325 andCPZ005 (Donnenberg et al. J. Infect. Dis., 169:831-838 (1994)).Attenuated Escherichia strains, such as the attenuated turkey pathogenE. coli 02 carAB mutant (Kwaga et al. Infect. Immun., 62:3766-3772(1994)) are preferably used in the present invention. Alternatively, newattenuated Escherichia strains can be constructed by introducing one ormore attenuating mutations in groups (i) to (vii) as described forShigella spp. above.

The particular Klebsiella strain employed is not critical to the presentinvention. Examples of Klebsiella strains which can be employed in thepresent invention include K pneumoniae (ATCC No. 13884). AttenuatedKlebsiella strains are preferably used in the present invention, and canbe constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Bordetella strain employed is not critical to the presentinvention. Examples of Bordetella strains which can be employed in thepresent invention include B. bronchiseptica (ATCC No. 19395). AttenuatedBordetella strains are preferably used in the present invention, and canbe constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Neisseria strain employed is not critical to the presentinvention. Examples of Neisseria strains which can be employed in thepresent invention include N. meningitidis (ATCC No. 13077) and N.gonorrhoeae (ATCC No. 19424). Attenuated Neisseria strains, such as N.gonorrhoeae MS 11 aro mutant (Chamberlain et al. Micro. Path., 15:51-63(1993)) are preferably used in the present invention. Alternatively, newattenuated Neisseria strains can be constructed by introducing one ormore attenuating mutations in groups (i) to (vii) as described forShigella spp. above.

The particular Aeromonas strain employed is not critical to the presentinvention. Examples of Aeromonas strains which can be employed in thepresent invention include A. eucrenophila (ATCC No. 23309).Alternatively, new attenuated Aeromonas strains can be constructed byintroducing one or more attenuating mutations in groups (i) to (vii) asdescribed for Shigella spp. above.

The particular Franciesella strain employed is not critical to thepresent invention. Examples of Franciesella strains which can beemployed in the present invention include F. tularensis (ATCC No.15482). Attenuated Franciesella strains are preferably used in thepresent invention, and can be constructed by introducing one or moreattenuating mutations in groups (i) to (vii) as described for Shigellaspp. above.

The particular Corynebacterium strain employed is not critical to thepresent invention. Examples of Corynebacterium strains which can beemployed in the present invention include C. pseudotuberculosis (ATCCNo. 19410). Attenuated Corynebacterium strains are preferably used inthe present invention, and can be constructed by introducing one or moreattenuating mutations in groups (i) to (vii) as described for Shigellaspp. above.

The particular Citrobacter strain employed is not critical to thepresent invention. Examples of Citrobacter strains which can be employedin the present invention include C. freundii (ATCC No. 8090). AttenuatedCitrobacter strains are preferably used in the present invention, andcan be constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Chlamydia strain employed is not critical to the presentinvention. Examples of Chlamydia strains which can be employed in thepresent invention include C. pneumoniae (ATCC No. VR1310). AttenuatedChlamydia strains are preferably used in the present invention, and canbe constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Hemophilus strain employed is not critical to the presentinvention. Examples of Hemophilus strains which can be employed in thepresent invention include H. sornnus (ATCC No. 43625). AttenuatedHemophilus strains are preferably used in the present invention, and canbe constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Brucella strain employed is not critical to the presentinvention. Examples of Brucella strains which can be employed in thepresent invention include B. abortus (ATCC No. 23448). AttenuatedBrucella strains are preferably used in the present invention, and canbe constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Mycobacterium strain employed is not critical to thepresent invention. Examples of Mycobacterium strains which can beemployed in the present invention include M. intracellulare (ATCC No.13950) and M. tuberculosis (ATCC No. 27294). Attenuated Mycobacteriumstrains are preferably used in the present invention, and can beconstructed by introducing one or more attenuating mutations in groups(i) to (vii) as described for Shigella spp. above.

The particular Legionella strain employed is not critical to the presentinvention. Examples of Legionella strains which can be employed in thepresent invention include L. pneumophila (ATCC No. 33156). AttenuatedLegionella strains, such as a L. pneumophila mip mutant (Ott, FEMSMicro. Rev., 14:161-176 (1994)) are preferably used in the presentinvention. Alternatively, new attenuated Legionella strains can beconstructed by introducing one or more attenuating mutations in groups(i) to (vii) as described for Shigella spp. above.

The particular Rhodococcus strain employed is not critical to thepresent invention. Examples of Rhodococcus strains which can be employedin the present invention include R. equi (ATCC No. 6939). AttenuatedRhodococcus strains are preferably used in the present invention, andcan be constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Pseudomonas strain employed is not critical to thepresent invention. Examples of Pseudomonas strains which can be employedin the present invention include P. aeruginosa (ATCC No. 23267).Attenuated Pseudomonas strains are preferably used in the presentinvention, and can be constructed by introducing one or more attenuatingmutations in groups (i) to (vii) as described for Shigella spp. above.

The particular Helicobacter strain employed is not critical to thepresent invention. Examples of Helicobacter strains which can beemployed in the present invention include H. mustelae (ATCC No. 43772).Attenuated Helicobacter strains are preferably used in the presentinvention, and can be constructed by introducing one or more attenuatingmutations in groups (i) to (vii) as described for Shigella spp. above.

The particular Salmonella strain employed is not critical to the presentinvention. Examples of Salmonella strains which can be employed in thepresent invention include Salmonella typhi (ATCC No. 7251) and S.typhimurium (ATCC No. 13311). Attenuated Salmonella strains arepreferably used in the present invention and include S. typhi aroC aroD(Hone et al. Vacc., 9:810-816 (1991)) and S. typhimurium aroA mutant(Mastroeni et al. Micro. Pathol, 13:477-491 (1992))). Alternatively, newattenuated Salmonella strains can be constructed by introducing one ormore attenuating mutations in groups (i) to (vii) as described forShigella spp. above.

The particular Vibrio strain employed is not critical to the presentinvention. Examples of Vibrio strains which can be employed in thepresent invention include Vibrio cholerae (ATCC No. 14035) and Vibriocincinnatiensis (ATCC No. 35912). Attenuated Vibrio strains arepreferably used in the present invention and include V. cholerae RSIvirulence mutant (Taylor et al. J. Infect. Dis., 170:1518-1523 (1994))and V. cholerae ctxA, ace, zot, cep mutant (Waldor et al. J. Infect.Dis., 170:278-283 (1994)). Alternatively, new attenuated Vibrio strainscan be constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Bacillus strain employed is not critical to the presentinvention. Examples of Bacillus strains which can be employed in thepresent invention include Bacillus subtilis (ATCC No. 6051). AttenuatedBacillus strains are preferably used in the present invention andinclude B. anthracis mutant pX01 (Welkos et al. Micro. Pathol,14:381-388 (1993)) and attenuated BCG strains (Stover et al. Nat.,351:456-460 (1991)). Alternatively, new attenuated Bacillus strains canbe constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Erysipelothrix strain employed is not critical to thepresent invention. Examples of Erysipelothrix strains which can beemployed in the present invention include Erysipelothrix rhusiopathiae(ATCC No. 19414) and Erysipelothrix tonsillarum (ATCC No. 43339).Attenuated Erysipelothrix strains are preferably used in the presentinvention and include E. rhusiopathiae Kg-1a and Kg-2 (Watarai et al. J.Vet. Med. Sci., 55:595-600 (1993)) and E. rhusiopathiae ORVAC mutant(Markowska-Daniel et al. Int. J. Med. Microb. Virol. Parisit. Infect.Dis., 277:547-553 (1992)). Alternatively, new attenuated Erysipelothrixstrains can be constructed by introducing one or more attenuatingmutations in groups (i) to (vii) as described for Shigella spp. above.

1.3. Methods for Increasing the Invasive Properties of a BacterialStrain

Whether organisms have been traditionally described as invasive ornon-invasive, these organisms can be engineered to increase theirinvasive properties, e.g., by mimicking the invasive properties ofShigella spp., Listeria spp., Rickettsia spp., or enteroinvasive E. colispp. For example, one or more genes that enable the microorganism toaccess the cytoplasm of a cell, e.g., a cell in the natural host of saidnon-invasive bacteria, can be introduced into the microorganism.

Examples of such genes referred to herein as “cytoplasm-targeting genes”include genes encoding the proteins that enable invasion by Shigella orthe analogous invasion genes of entero-invasive Escherichia, orlisteriolysin O of Listeria, as such techniques are known to result inrendering a wide array of invasive bacteria capable of invading andentering the cytoplasm of animal cells (Formal et al. Infect. Immun.,46:465 (1984); Bielecke et al. Nature, 345:175-176 (1990); Small et al.In: Microbiology-1986, pages 121-124, Levine et al. Eds., AmericanSociety for Microbiology, Washington, D. C. (1986); Zychlinsky et al.Molec. Micro., 11:619-627 (1994); Gentschev et al. (1995) Infection &Immunity 63:4202; Isberg, R. R. and S. Falkow (1985) Nature 317:262; andIsberg, R. R. et al. (1987) Cell 50:769). Methods for transferring theabove cytoplasm-targeting genes into a bacterial strain are well knownin the art. Another preferred gene which can be introduced into bacteriato increase their invasive character encodes the invasin protein fromYersinia pseudotuberculosis, (Leong et al. EMBO J., 9:1979 (1990)).Invasin can also be introduced in combination with listeriolysin,thereby further increasing the invasive character of the bacteriarelative to the introduction of either of these genes. The above geneshave been described for illustrative purposes; however, it will beobvious to those skilled in the art that any gene or combination ofgenes, from one or more sources, that participates in the delivery of amolecule, in particular an RNA or RNA-encoding DNA molecule, from amicroorganism into the cytoplasm of a cell, e.g., an animal cell, willsuffice. Thus, such genes are not limited to bacterial genes, andinclude viral genes, such as influenza virus hemagglutinin HA-2 whichpromotes endosmolysis (Plank et al. J. Biol. Chem., 269:12918-12924(1994)).

The above cytoplasm-targeting genes can be obtained by, e.g., PCRamplification from DNA isolated from an invasive bacterium carrying thedesired cytoplasm-targeting gene. Primers for PCR can be designed fromthe nucleotide sequences available in the art, e.g., in the above-listedreferences and/or in GenBank, which is publicly available on theinternet (www.ncbi.nlm nih.gov/). The PCR primers can be designed toamplify a cytoplasm-targeting gene, a cytoplasm-targeting operon, acluster of cytoplasm-targeting genes, or a regulon ofcytoplasm-targeting genes. The PCR strategy employed will depend on thegenetic organization of the cytoplasm-targeting gene or genes in thetarget invasive bacteria. The PCR primers are designed to contain asequence that is homologous to DNA sequences at the beginning and end ofthe target DNA sequence. The cytoplasm-targeting genes can then beintroduced into the target bacterial strain, e.g., by using Hfr transferor plasmid mobilization (Miller, A Short Course in Bacterial Genetics,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1992);Bothwell et al. supra; and Ausubel et al. supra), bacteriophage-mediatedtransduction (de Boer, supra; Miller, supra; and Ausubel et al. supra),chemical transformation (Bothwell et al. supra; Ausubel et al. supra),electroporation (Bothwell et al. supra; Ausubel et al. supra; andSambrook, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) and physical transformationtechniques (Johnston et al. supra; and Bothwell, supra). Thecytoplasm-targeting genes can be incorporated into lysogenicbacteriophage (de Boer et al. Cell, 56:641-649 (1989)), plasmids vectors(Curtiss et al. supra) or spliced into the chromosome (Hone et al.supra) of the target strain.

In addition to genetically engineering bacteria to increase theirinvasive properties, as set forth above, bacteria can also be modifiedby linking an invasion factor to the bacteria. Accordingly, in oneembodiment, a bacterium is rendered more invasive by coating thebacterium, either covalently or non-covalently, with an invasion factor,e.g., the protein invasin, invasin derivatives, or a fragment thereofsufficient for invasiveness. In fact, it has been shown thatnon-invasive bacterial cells coated with purified invasin from Yersiniapseudotuberculosis or the carboxyl-terminal 192 amino acids of invasinare able to enter mammalian cells (Leong et al. (1990) EMBO J. 9:1979).Furthermore, latex beads coated with the carboxyl terminal region ofinvasin are efficiently internalized by mammalian cells, as are strainsof Staphylococcus aureus coated with antibody-immobilized invasin(reviewed in Isberg and Tran van Nhieu (1994) Ann. Rev. Genet. 27:395).Alternatively, a bacterium can also be coated with an antibody, variantthereof, or fragment thereof which binds specifically to a surfacemolecule recognized by a bacterial entry factor. For example, it hasbeen shown that bacteria are internalized if they are coated with amonoclonal antibody directed against an integrin molecule, e.g., α5β1,known to be the surface molecule with which the bacterial invasinprotein interacts (Isberg and Tran van Nhieu, supra). Such antibodiescan be prepared according to methods known in the art. The antibodiescan be tested for efficacy in mediating bacterial invasiveness by, e.g.,coating bacteria with the antibody, contacting the bacteria witheukaryotic cells having a surface receptor recognized by the antibody,and monitoring the presence of intracellular bacteria, according to themethods described above. Methods for linking an invasion factor to thesurface of a bacterium are known in the art and include cross-linking.

2. Target Cells

The invention provides a method for delivering RNA to any type of targetcell. As used herein, the term “target cell” refers to a cell which canbe invaded by a bacterium, i.e., a cell which has the necessary surfacereceptor for recognition by the bacterium.

Preferred target cells are eukaryotic cells. Even more preferred targetcells are animal cells. “Animal cells” are defined as nucleated,non-chloroplast containing cells derived from or present inmulticellular organisms whose taxanomic position lies within the kingdomanimalia. The cells may be present in the intact animal, a primary cellculture, explant culture or a transformed cell line. The particulartissue source of the cells is not critical to the present invention.

The recipient animal cells employed in the present invention are notcritical thereto and include cells present in or derived from allorganisms within the kingdom animalia, such as those of the familiesmammalia, pisces, avian, reptilia.

Preferred animal cells are mammalian cells, such as humans, bovine,ovine, porcine, feline, canine, goat, equine, and primate cells. Themost preferred animal cells are human cells.

In a preferred embodiment, the target cell is in a mucosal surface.Certain enteric pathogens, e.g., E. coli, Shigella, Listeria, andSalmonella, are naturally adapted for this application, as theseorganisms possess the ability to attach to and invade host mucosalsurfaces (Kreig et al. supra). Therefore, in the present invention, suchbacteria can deliver RNA molecules or RNA-encoding DNA to cells in thehost mucosal compartment.

Although certain types of bacteria may have a certain tropism, i.e.,preferred target cells, delivery of RNA or RNA-encoding DNA to a certaintype of cell can be achieved by choosing a bacterium which has a tropismfor the desired cell type or which is modified such as to be able toinvade the desired cell type. Thus, e.g., a bacterium could begenetically engineered to mimic mucosal tissue tropism and invasiveproperties, as discussed above, to thereby allow said bacteria to invademucosal tissue, and deliver RNA or RNA-encoding DNA to cells in thosesites.

Bacteria can also be targeted to other types of cells. For example,bacteria can be targeted to erythrocytes of humans and primates bymodifying bacteria to express on their surface either, or both of, thePlasmodium vivax reticulocyte binding proteins-1 and -2, which bindspecifically to erythrocytes in humans and primates (Galinski et al.Cell, 69:1213-1226 (1992)). In another embodiment, bacteria are modifiedto have on their surface asialoorosomucoid, which is a ligand for theasilogycoprotein receptor on hepatocytes (Wu et al. J. Biol. Chem.,263:14621-14624 (1988)). In yet another embodiment, bacteria are coatedwith insulin-poly-L-lysine, which has been shown to target plasmiduptake to cells with an insulin receptor (Rosenkranz et al. Expt. CellRes., 199:323-329 (1992)). Also within the scope of the invention arebacteria modified to have on their surface p60 of Listeriamonocytogenes, which allows for tropism for hepatocytes (Hess et al.Infect. Immun., 63:2047-2053 (1995)), or a 60 kD surface protein fromTrypanosoma cruzi which causes specific binding to the mammalianextra-cellular matrix by binding to heparin, heparin sulfate andcollagen (Ortega-Barria et al. Cell, 67:411-421 (1991)).

Yet in another embodiment, a cell can be modified to become a targetcell of a bacterium for delivery of RNA. Accordingly, a cell can bemodified to express a surface antigen which is recognized by a bacteriumfor its entry into the cell, i.e., a receptor of an invasion factor. Thecell can be modified either by introducing into the cell a nucleic acidencoding a receptor of an invasion factor, such that the surface antigenis expressed in the desired conditions. Alternatively, the cell can becoated with a receptor of an invasion factor. Receptors of invasionfactors include proteins belonging to the integrin receptor superfamily.A list of the type of integrin receptors recognized by various bacteriaand other microorganisms can be found, e.g., in Isberg and Tran VanNhieu (1994) Ann. Rev. Genet. 27:395. Nucleotide sequences for theintegrin subunits can be found, e.g., in GenBank, publicly available onthe internet.

As set forth above, yet other target cells include fish, avian, andreptilian cells. Examples of bacteria which are naturally invasive forfish, avian, and reptilian cells are set forth below.

Examples of bacteria which can naturally access the cytoplasm of fishcells include, but are not limited to Aeromonas salminocida (ATCC No.33658) and Aeromonas schuberii (ATCC No. 43700). Attenuated bacteria arepreferably used in the invention, and include A. salmonicidia vapA(Gustafson et al. J. Mol. Biol., 237:452-463 (1994)) or A. salmonicidiaaromatic-dependent mutant (Vaughan et al. Infect. Immun., 61:2172-2181(1993)).

Examples of bacteria which can naturally access the cytoplasm of aviancells include, but are not restricted to, Salmonella galinarum (ATCC No.9184), Salmonella enteriditis (ATCC No. 4931) and Salmonella typhimurium(ATCC No. 6994). Attenuated bacteria are preferred to the invention andinclude attenuated Salmonella strains such as S. galinarum cya crpmutant (Curtiss et al. (1987) supra) or S. enteritidis aroAaromatic-dependent mutant CVL30 (Cooper et al. Infect. Immun.,62:4739-4746 (1994)).

Examples of bacteria which can naturally access the cytoplasm ofreptilian cells include, but are not restricted to, Salmonellatyphimurium (ATCC No. 6994). Attenuated bacteria are preferable to theinvention and include, attenuated strains such as S. typhimuirumaromatic-dependent mutant (Hormaeche et al. supra).

The invention also provides for delivery of RNA to other eukaryoticcells, e.g., plant cells, so long as there are microorganisms which arecapable of invading such cells, either naturally or after having beenmodified to become invasive. Examples of microorganisms which can invadeplant cells include Agrobacterium tumerfacium, which uses a pilus-likestructure which binds to the plant cell via specific receptors, and thenthrough a process that resembles bacterial conjugation, delivers atleast some of its content to the plant cell.

Set forth below are examples of cell lines to which RNA can be deliveredaccording to the method of this invention.

Examples of human cell lines include but are not limited to ATCC Nos.CCL 62, CCL 159, HTB 151, HTB 22, CCL 2, CRL 1634, CRL 8155, HTB 61, andHTB104.

Examples of bovine cell lines include ATCC Nos. CRL 6021, CRL 1733, CRL6033, CRL 6023, CCL 44 and CRL 1390.

Examples of ovine cells lines include ATCC Nos. CRL 6540, CRL 6538, CRL6548 and CRL 6546.

Examples of porcine cell lines include ATCC Nos. CL 184, CRL 6492, andCRL 1746.

Examples of feline cell lines include CRL 6077, CRL 6113, CRL 6140, CRL6164, CCL 94, CCL 150, CRL 6075 and CRL 6123.

Examples of buffalo cell lines include CCL 40 and CRL 6072.

Examples of canine cells include ATCC Nos. CRL 6213, CCL 34, CRL 6202,CRL 6225, CRL 6215, CRL 6203 and CRL 6575.

Examples of goat derived cell lines include ATCC No. CCL 73 and ATCC No.CRL 6270.

Examples of horse derived cell lines include ATCC Nos. CCL 57 and CRL6583.

Examples of deer cell lines include ATCC Nos. CRL 6193-6196.

Examples of primate derived cell lines include those from chimpanzee'ssuch as ATCC Nos. CRL 6312, CRL 6304, and CRL 1868; monkey cell linessuch as ATCC Nos. CRL 1576, CCL 26, and CCL 161; orangautan cell lineATCC No. CRL 1850; and gorilla cell line ATCC No. CRL 1854.

4. Pharmaceutical Compositions

In a preferred embodiment of the invention, the invasive bacteriacontaining the RNA molecules, and/or DNA encoding such, are introducedinto an animal by intravenous, intramuscular, intradermal,intraperitoneally, peroral, intranasal, intraocular, intrarectal,intravaginal, intraosseous, oral, immersion and intraurethralinoculation routes.

The amount of the live invasive bacteria of the present invention to beadministered to a subject will vary depending on the species of thesubject, as well as the disease or condition that is being treated.Generally, the dosage employed will be about 10³ to 10¹¹ viableorganisms, preferably about 10⁵ to 10⁹ viable organisms per subject.

The invasive bacteria of the present invention are generallyadministered along with a pharmaceutically acceptable carrier and/ordiluent. The particular pharmaceutically acceptable carrier an/ordiluent employed is not critical to the present invention. Examples ofdiluents include a phosphate buffered saline, buffer for bufferingagainst gastric acid in the stomach, such as citrate buffer (pH 7.0)containing sucrose, bicarbonate buffer (pH 7.0) alone (Levine et al. J.Clin. Invest., 79:888-902 (1987); and Black et al J. Infect. Dis.,155:1260-1265 (1987)), or bicarbonate buffer (pH 7.0) containingascorbic acid, lactose, and optionally aspartame (Levine et al. Lancet,11:467-470 (1988)). Examples of carriers include proteins, e.g., asfound in skim milk, sugars, e.g., sucrose, or polyvinylpyrrolidone.Typically these carriers would be used at a concentration of about0.1-30% (w/v) but preferably at a range of 1-10% (w/v).

Set forth below are other pharmaceutically acceptable carriers ordiluents which may be used for delivery specific routes. Any suchcarrier or diluent can be used for administration of the bacteria of theinvention, so long as the bacteria are still capable of invading atarget cell. In vitro or in vivo tests for invasiveness can be performedto determine appropriate diluents and carriers. The compositions of theinvention can be formulated for a variety of types of administration,including systemic and topical or localized administration. Lyophilizedforms are also included, so long as the bacteria are invasive uponcontact with a target cell or upon administration to the subject.Techniques and formulations generally may be found in Remmington'sPharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemicadministration, injection is preferred, including intramuscular,intravenous, intraperitoneal, and subcutaneous. For injection, thecomposition, e.g., bacteria, of the invention can be formulated inliquid solutions, preferably in physiologically compatible buffers suchas Hank's solution or Ringer's solution.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner.

For administration by inhalation, the pharmaceutical compositions foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the composition, e.g., bacteria, and asuitable powder base such as lactose or starch.

The pharmaceutical compositions may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The pharmaceutical compositions may also be formulated in rectal,intravaginal or intraurethral compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the bacteria of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art, so long as the bacteria are still invasiveupon contact with a target cell.

The compositions may, if desired, be presented in a pack or dispenserdevice and/or a kit which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

The invasive bacteria containing the RNA or RNA-encoding DNA to beintroduced can be used to infect animal cells that are cultured invitro, such as cells obtained from a subject. These in vitro-infectedcells can then be introduced into animals, e.g., the subject from whichthe cells were obtained initially, intravenously, intramuscularly,intradermally, or intraperitoneally, or by any inoculation route thatallows the cells to enter the host tissue. When delivering RNA toindividual cells, the dosage of viable organisms to administered will beat a multiplicity of infection ranging from about 0.1 to 10⁶, preferablyabout 10² to 10⁴ bacteria per cell.

In yet another embodiment of the present invention, bacteria can alsodeliver RNA molecules encoding proteins to cells, e.g., animal cells,from which the proteins can later be harvested or purified. For example,a protein can be produced in a tissue culture cell.

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references including literature references, issued patents,published patent applications as cited throughout this application arehereby expressly incorporated by reference. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, AlanR. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

EXAMPLES Methods

siRNA-Generating Plasmid Construction:

Oligonucleotides were obtained at 0.2 μmol from QIAGEN with PAGEpurification. After annealing, oligonucleotides were inserted into theBamHI and HindIII binding sites within pSilencer 2.0-U6 (Ambion, Inc.)according to the manufacturer's instructions.

The following sequences were used:

The k-Ras-1 and -2 64-mers target the nucleotides encoding for aminoacids 9-15 of k-Ras protein which spans the specific mutation of V12.

k-Ras-1 (64-mer): (SEQ ID NO: 1)5′gATCCCgTTggAgCTgTTggCgTAgTTCAAgAgACTACgCCAACAgCT CCAACTTTTTTggAAA3′k-Ras-2 (64-mer): (SEQ ID NO: 2)5′AgCTTTTCCAAAAAAgTTggAgCTgTTggCgTAgTCTCTTgAACTACg CCAACAgCTCCAACgg3′

The β-Catenin-1 and -2 64-mers target the nucleotides encoding for aminoacids 79-85 within the catenin protein

β-Catenin-1 (64mer): (SEQ ID NO:3)5′gATCCCAgCTgATATTgATggACAgTTCAAgAgACTgTCCATCAATAT CAgCTTTTTTTggAAA3′β-Catenin-2 (64mer): (SEQ ID NO: 4)5′AgCTTTTCCAAAAAAAgCTgATATTgATggACAgTCTCTTgAACTgTC CATCAATATCAgCTgg3′

The EGFP-1 and -2 64-mers target the nucleotides encoding for aminoacids 22-28 of EGFP.

EGFP-1 (64-mer): (SEQ ID NO: 5)5′gATCCCgACgTAAACggCCACAAgTTTCAAgAgAACTTgTggCCgTTT ACgTCTTTTTTggAAA3′EGFP-2 (64-mer): (SEQ ID NO: 6)5′AgCTTTTCCAAAAAAgACgTAAACggCCACAAgTTCTCTTgAAACTTg TggCCgTTTACgTCgg3′

Transkingdom RNA Interference Plasmid Construction:

The engineered plasmid pT7RNAi-Hly-Inv, TRIP was constructed from pGB2Ωinv-hly (Milligan et al., Nucleic Acids Res. 15, 8783 (1987)) andpBlueScript II KS(+). Oligonucleotides containing multiple cloning site(MCS), T7 promoter, enhancer and terminator (synthesized from Qiagen)were ligated into blunted BssHII sites of KSII(+), and β-catenin hairpinoligos were inserted into BamHI and SalI sites of MCS to generateplasmid pT7RNAi. PstI fragments containing the inv locus of pGB2Ωinv-hly were inserted into PstI site of KSII(+). Using pGB2 Ωinv-hly astemplate, HlyA gene was amplified by PCR (Pfx DNA polymerase, InvitrogenInc.) with primers, hly-1: 5′-CCCTCCTTTGATTAGTATATTCCTATCTTA-3′ (SEQ IDNO:7) and hly-2: 5′-AAGCTTTTAAATCAGCAGGGGTCTTTTTGG-3′ (SEQ ID NO:8), andwere cloned into EcoRV site of KSII(+)/Inv. Hly-Inv fragment was excisedwith BamHI and SalI. After blunting, it was ligated into EcoRV siteincorporated within T7 terminator of pT7RNAi Bacteria:

The auxotrophic Salmonella typhimurium aroA 7207 (S. typhimurium 2337-65derivative hisG46, DEL407[aroA544::Tn10(Tc-s)] used as the plasmidcarrier in this study was kindly provided by Prof. BAD Stocker, StanfordUniversity, CA. Escherichia coli XL-1 Blue was used to maintain theplasmids (Strategene).

Transformation of SL 7207 was achieved using an adapted electroporationprotocol (1). Competent SL7207 and 1 μg plasmid were incubated on ice ina chilled 0.2 cm electroporation cuvette for 5 min. A 2.5 kV, 25 μF,200Ω impulse was applied using a BioRad Genepulser. 1 mL of prewarmedSOC medium was added and bacteria were allowed to recover for 1 hr at37° C. with 225 RPM shaking before plating on selective agar plates.Presence of the plasmids was confirmed using minipreparation afteralcalinic lysis and separation on 0.7% agarose gel.

For in vitro experiments, SL 7202 were grown overnight at 37° C. inLuria Broth (LB) supplemented with 100 μg/mL Ampicillin (for SL-siRAS,SL-siGFP and SL-siCAT) without shaking. The next morning, bacteria weregrown in fresh medium after 1% inoculation from the overnight cultureuntil reaching an OD₆₀₀ of 0.4-0.6. Bacteria were centrifuged (3500 RPM,4° C.) washed once in phosphate-buffered saline (PBS) and resuspended inPBS at the desired concentrations. For all determinations of bacterialnumber and concentration, the bacterial density was measuredspectrometrically and calculated according to the formulac=OD₆₀₀*8×10⁸/mL.

For animal experiments, SL 7207 were grown in Brain Heart Infusion Broth(Sigma) in a stable culture overnight supplemented with the appropriateantibiotics where required. Bacteria were washed and resuspended in PBSat a concentration of 2.5×10¹⁰/mL. Serial dilutions were done and platedon selective agar at several times during the experiment to verify theactual number of bacteria administered.

Plasmids were also transformed into BL21DE3 strain (Gene TherapySystems) according to the manufacturer instructions. Bacteria were grownat 37° C. in Brain-Heart-Infusion-broth with addition of 100 μg/mlAmpicillin. Bacteria numbers were calculated using OD₆₀₀ measurement.For cell infection, overnight cultures were inoculated into fresh mediumfor another 2 h growth.

Cell Culture:

A human colon cancer cell line (SW 480) was used herein. It carries amutation of APC protein resulting in increased basal levels ofβ-catenin. A stably GFP-expressing cell line derived from yolk sacepithelium, CRL 2583 (ATCC, Rockville, Md.) was used for GFP-knockdownexperiments. CRL 2583 was maintained in 200 μg/mL G418 until 30 minbefore bacterial infection. SW 480 were grown in RPMI-1640 supplementedwith 10% fetal bovine serum. CRL 2583 were grown in high glucose, highNaHCO₃ DMEM supplemented with 15% FBS as recommended by the supplier.All growth media were routinely supplemented with antibiotics: 100 U/mlpenicillin G, 10 μg/ml streptomycin, 2.5 μg/ml amphotericin (all mediaand additives purchased from Sigma, St. Louis).

For direct transfection of plasmids, 500,000 cells were seeded into 6 cmpetri dishes and allowed to grow overnight before they were transfectedusing a standard CaP-coprecipitation protocol.

Briefly, 15 μg plasmid-DNA are mixed in 500 μL reaction mix (2×HEPESbuffer, 60 μL CaP) and dropped to the cells in fresh medium without FBS.Precipitation was allowed to continue for 9 hrs before precipitates werewashed away. Cells were harvested at different time points (36, 48, 60,72, and 96 hrs).

For standard bacterial infection assays, 500,000 cells were seeded into6 cm petri dishes and were allowed to attach overnight. 30 min prior toaddition of the bacteria, the growth medium was replaced with freshmedium without antibiotics and fetal bovine serum. SL 7207-siRAS,-siCAT, -siGFP were added in 500 μL PBS to reach the designatedmultiplicity of infection (MOI) of 100, 500 or 1000 and infection wascarried out in a standard incubator with 37° C., 5% CO₂. By the end ofthe indicated infection period, plates were washed once with 4 ml ofserum-free RPMI medium and 3 times with 4 ml PBS, then 5 ml of freshcomplete RPMI medium containing 100 μg/mL of ampicillin and 150 μg/mL ofgentamycin were added. Twenty-four hours later, tetracycline was addedto final concentration of 15 μg/mL. At indicated different time points(24-96 h) after bacterial invasion, cells were harvested for westernblot or flow cytometry.

For staining of intracellular bacteria, cells were grown on Lab-Tek IIChamber Slides (Nalgene Nunc, USA). After bacterial invasion asdescribed above, cells were washed with PBS and fixed in 1%paraformaldeyde for 10 min. Acridin Orange (Sigma) solution (0.01%) wasadded for 45 sec, then washed with PBS. Crystal Violet stain (Sigma) wasapplied for 45 sec, then washed with PBS. Coverslips were mounted usingPERMOUNT™ mounting medium and invasion was assessed using confocalmicroscopy.

MTT Assay:

After treatment with SL7207-siRAS and/or SL7207-siCAT, cells weretrypsinized (24 h or 48 h later), diluted and seeded into 96-well plateat a concentration of 5000 cells/well. Cells were then allowed to growfor up to 4 days. At the desired incubation time point, medium wasremoved and 100 μl of MTT solution (5 mg/mL) was added to each well.After an incubation period of 4 h, MTT solution was drained away andcells were lysed by adding 100 μL of solubilization reagent(Isopropanol:1N HCl:10% SDS 43:2:5) to each well. The resulting signalof the dark blue formazan-product was photometrically determined at 570nm wavelength. The amount of color formation is dependent on the numberof surviving cells per well.

Colony Formation Test:

After treatment with SL7207-siRAS and/or SL7207-siCAT, cells weretrypsinized (24 h post-transfection), diluted and seeded into 6-well MTPat a concentration of 750 cells/well. Cells were kept growing for twomore weeks to let them form visible colonies. Two weeks later, mediumwas removed and 1 ml of Giemsa stain (7.415 g/L) were added to eachwell. After 10-min incubation at 37° C., Giemsa stain was drained awayand cells washed with PBS. Groups of more than eight cells were countedas positive colonies.

Western Blot:

Cells were washed with chilled PBS, scraped off and lysed in lysisbuffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1%NP-40, 1 mM DTT) containing 0.1% protease inhibitor mix (Sigma). 20 μgof protein were separated using 11% SDS-Page Gel and transferred to a0.4 μm PVDF membrane (Schleicher and Schuell). The membrane was blockedusing 5% milk and incubated for 1 hr with specific antibodies at theindicated concentrations: Living Colors® antibody (Clontech)-1:500,β-catenin antibody (Santa Cruz)-1:500, k-Ras antibody (Santa Cruz)-1:300and β-actin (Santa Cruz) 1:500. Each was followed by incubation withhorseradish-peroxidase conjugated anti-rabbit or anti-goat secondaryantibodies (Santa Cruz)-1:1000-1:2000. Bands were detected using ECL®chemoluminescence detection (Amersham).

Flow Cytometry:

For flow cytometry, cells were trypsinized for 3 min, resuspended infresh medium and washed in PBS. After centrifugation, cells were fixedfor 10 min in 1% paraformaldehyde/PBS at 4° C. Flow cytometry wasperformed using FACScan (Becton Dickinson), data analysis was done usingCellQuest® software.

Animal Techniques:

Six to eight week old female GFP+ transgenic mice (CgTg5Nagy) wereobtained from Jackson laboratories. They were housed in the BIDMC animalresearch facility with ad libitum access to standard rodent diet anddrinking water. Treatment was initiated at ten weeks of age. For the ivtreatment protocol, four doses of 10⁶ cfu SL-siRAS or SL-siGFP dissolvedin 50 μL PBS were injected into the tail vein on alternating days. Micewere weighed daily and monitored for signs of disease.

Mice were sacrificed one day after the final treatment at which timetissue samples were taken for histochemistry and flow cytometricanalysis. Tissues were paraffin embedded and sectioned in 6 μm steps forhistochemistry and fluorescence microscopy. For flow cytometry,hepatocyte and splenocyte suspensions were prepared through the use ofcell strainers (Falcon). Organ suspensions were fixed in 4% formalin andflow cytometry was performed using FACScan (Becton Dickinson), dataanalysis was done using CellQuest® software.

For the Xenograft cancer model, female BALB/c nude mice (Charles RiverLaboratories) were randomized into two groups (n=6). Three weeks beforetreatment, 1×10⁷ of SW480 cells were implanted subcutaneously.Treatments were initiated when the tumors reached about 10 mm indiameter. The treatment group was injected through tail vein with 1×10⁸cfu of E. coli expressing shRNA against β-catenin in PBS. The controlgroup was similarly treated except that the E. coli contains the TRIPvector without shRNA insert. The treatment was carried out every 5 daysfor a total of three treatments. Mice were sacrificed 5 days after thelast treatment. Tissues were frozen and fixed for analysis of β-cateninmRNA level by real-time PCR and β-catenin protein level byimmunohistochemistry.

For in vivo silencing experiments, female C57/BL6 mice (Charles RiverLaboratories) were randomly divided into two groups. The treatment groupwas administered orally with 5×10¹⁰ cfu E. coli expressing shRNA againstβ-catenin in 200 μL phosphate-buffered saline (PBS). The control groupwas similarly treated except that the E. coli contains the TRIP vectorwithout the shRNA insert. Two independent experiments were performedwith 6 and 5 mice per group used, respectively. The treatment wascarried out daily for 5 days per week for a total of 4 weeks. Mice weresacrificed 2 days after the last treatment, and tissues wereparaffin-embedded.

Immunhistochemistry

Immunostaining was performed on 6 μm tissue sections using VectastainElite ABC avidin-biotin staining kit (Vector laboratories, Burlingame,Calif.) according to the instructions by the manufacturer. Slides weredeparaffinized and rehydrated using a standard protocol. For antigenretrieval, slides were heated by microwave in 5% urea for 5 min.Unspecific binding sites were blocked with 1% bovine serum albumin for10 min and endogenous peroxidase activity was suppressed by treatmentwith 3% H₂O₂ in methanol for 10 min. Sections were exposed to primaryantibody LIVING COLORS™ rabbit polyclonal antibody (Clontech) at 1:500dilution overnight at 4° C. The chromogen used was 3,3′Diamino-enzidine(DAB) (Vector), counterstaining was done with hematoxyline.

Interferon Response Detection:

SW480 cells were treated for 2 h with E. coli transformed with the TRIPencoding shRNA against human β-catenin or mutant k-Ras at MOI of 1:1000.Untreated cells were used as control. Cells were harvested at 24, 48 and72 h. The expression levels of OAS1, OAS2, MX1, ISGF3γ and IFITM1 geneswere determined by RT-PCR using the Interferon Response Detection Kit(SBI System Biosciences, CA).

Example 1 Knock Down of Green Fluorescent Protein Using BacteriaMediated Gene Silencing In Vitro and In Vivo

In the following experiments, an attenuated strain of Salmonellatyphimurium (SL 7207, obtained from BAD Stocker, Stanford University)was used. To prove that the concept is useful as a general approach, wedid confirmation experiments also with another attenuated strain ofSalmonella typhimurium (VNP 70009, obtained from VION Pharmaceuticals,New Haven) and an invasive and attenuated strain of E. coli (BM 2710,obtained from P. Courvalin, Institut Pasteur, Paris).

Silencing plasmids were designed based on a commercially availableplasmid (pSilencer, Ambion) to knock down the target genes GFP,β-catenin and oncogenic k-Ras (V12G). These plasmids were transformedinto SL 7207 by electroporation and positive clones were verified bygrowth on selective agar and DNA preparation.

For in vitro use, knockdown of GFP expression was demonstrated using thestable GFP+ cell line CRL 2598 (ATCC, Rockland, Va.). Knockdown ofoncogenic k-Ras (V12G) and β-catenin was demonstrated using the coloncancer cell line SW 480 and the pancreatic cancer cell line CAPAN-1.

A system of bacterial delivery using an invasive bacterial strain, S.typhimurium, was developed with a commercially available eukaryotictranscription plasmid, pSilencer (Ambion). The S. typhimurium strain SL7207 (kindly provided by B. Stocker, Stanford University) is attenuatedthrough an auxotrophy in the synthesis pathway for aromatic amino acids,and dies quickly after invasion into a target cell due to lack ofnutrients. This strain has been used successfully for delivery of DNA invitro and in mouse models, mainly with the purpose of DNA vaccination.

To verify bacterial entry into epithelial cells, an invasion assay wasperformed. SW 480 cells were infected for 2 hrs with SL-siRAS followedby 2 hrs of treatment with gentamycin. Acridin orange/crystal violetstaining revealed good invasion efficiency. 90% of the SW 480 cellsharbored viable SL 7207 bacteria. The average number of intracellularbacteria was 6 (range, 2-8) (FIG. 1). (Micrograph A1 is the transmissionimage. Micrograph A2 is the fluorescent image. Micrograph A3 is themerged image.) The number of viable intracellular bacteria reducedquickly over time. After 24 hrs and 48 hrs, only 10% and 3% of cellswere found to still contain bacteria.

In the next experiment, The effective reduction of GFP expression in theGFP+ cell line was demonstrated. Successful knockdown of oncogenes k-Rasand β-catenin was confirmed using Western blot and RT-PCR. Oncogeneknockdown resulted in growth retardation and decreased tumor formationin a xenograft animal model.

Cells stably expressing GFP (CRL 2583) were infected with SL7207carrying pSilencer2.0 including a sequence to silence GFP mRNA(SL-siGFP). (See above). After 48 hrs, cells treated with SL-siGFPshowed a marked decrease in GFP expression as compared to cells treatedwith SL-siRAS and untreated control (FIGS. 1B and 1C). Treatment withSL-siGFP led to loss of GFP signal in a manner dependent on themultiplicity of infection (MOI) applied. In untreated cells, only 4.3%display low or absent fluorescence. In SL-siGFP treated cells, thisfraction increased to 78.1% (treated with MOI 1:500) and 92.3% (MOI1:1000). Control treatment with SL-siRAS lead to a slight loss offluorescence (7.5% at MOI 1:500 and 8.4% at MOI 1:1000). This is alsoshown in the fluorescence microscope photograph in FIG. 1C. The topmicrograph is (200×) of SL-siRAS and the bottom of SL-siGFP (below)treated CRL 2583 cells. This finding was confirmed using flow cytometry.

In a series of animal experiments with stably GFP-expressing mice, wewere able to demonstrate knockdown of GFP expression in the liver and inthe colon (in both organs approx 50% reduction of GFP expression) afteroral and intravenous application of SL 7207 carrying the GFP-silencingplasmid.

In animal experiments, S. typhimurium was used to achieve gene silencingin a transgenic mouse model (GFP+). Using this method, silencing of thetransgene in the animal experiment is demonstrated at mRNA level as wellas on protein levels and tissue sections of various organs (liver,gastrointestinal tract) with limited toxicity.

Example 2 Knock Down of k-Ras and β-Catenin Using Bacteria Mediated GeneSilencing

Next, BMGS was applied to knock down a specific disease-related gene.The specific oncogenic point mutation in the k-Ras gene, k-Ras^(V12G),which is present in the human colon carcinoma cell line, SW 480 wastargeted.

After construction of the silencing plasmids and before they wereelectroporated into the attenuated SL7207, their activity was tested bytransfecting them into SW 480 cells using CaP coprecipitation.

Western blot (FIG. 4A) shows efficient knockdown of k-Ras using thepSilencer-kras (V12G) insert at 36 h and 48 h posttransfection. At latertime points, the protein expression recovers, which is due to outgrowthof transfected clones which have a growth disadvantage versus nontransfected clones in which the oncogenic k-Ras would still drivereplication. When BMGS with SL7207 as a carrier was used to mediate theknockdown, k-Ras levels were decreased at MOI of 1:500 and 1:1000. UsingBMGS, knockdown of the k-Ras protein was observed with similarefficiency compared to direct transfection of the silencer plasmid usingcalcium-phosphate coprecipitation, although the onset of knockdown wasslightly delayed by 12 hrs. (FIG. 4A). With an MOI of 1:1000, the resultcan be observed for a longer time (up to 72 hrs) (FIG. 2A).

The Western blot for β-catenin (FIG. 4B) shows delayed knockdown aftertransfection, with a maximum effect seen at 96 hrs post transfection. Itis assumed that this delay is caused by the survival time of SL 7207intracellularly before the plasmid is liberated (FIG. 2A).

After treatment with SL-siRAS and resulting knockdown of the oncogenick-Ras (V12G), SW 480 cells displayed significantly reduced viability andcolony formation ability (FIG. 2B). Cells were coincubated with equalamounts (2.5×10⁸) of SL-siRAS and SL-siCAT bacteria. Control cells weretreated with untransformed SL 207. 48 hrs after transfection, cells wereseeded in 96 well plates for MTT test and 6 well plates for colonyformation assays. At 120 hrs after treatment, viability, as assessed byMTT assay, was reduced to 62.5% after SL-siRAS treatment and 51% afterSL-siCAT treatment. Combined treatment further reduced viability to 29%of control treated cells. SL-siRAS treatment and SL-siCAT treatmentreduced the ability of SW 480 to form colonies by 37.7% and 50%,respectively. Combined treatment lead to 63.3% reduction.

Further, treatment with SL-RAS completely inhibited the tumor formationability of SW 480 cells when injected subcutaneously into nude mice,while treatment with empty SL 7207 did not influence their ability toform tumors (FIG. 2C). SW 480 cells (untreated, treated with SL 7207 orSL-siRAS) were subcutaneously injected into nude mice (4×10⁶ cells, n=4animals per group). Pretreatment with SL-siRAS completely abolished theability to form tumors (no tumors visible in any of the four animals,day 40) (FIG. 2C).

To test whether this approach can be employed universally, anothercancer-related gene, β-catenin was targeted (FIG. 2A). Basal levels ofβ-catenin are high in SW 480 cells, due to a mutated APC-gene, but canbe reduced through treatment with hairpin siRNA after pSilencer(siCAT)transfection (FIG. 2A). After treatment with SL 7207 carrying pSilencerwith the β-catenin construct (SL-siCAT), significant knockdown ofβ-catenin expression was achieved which resulted in decreased viabilityand colony formation ability (FIG. 2A). β-catenin was knocked down from96 hr, but recovered from 144 hr.

Example 3 In Vivo Bacterial Mediated Gene Silencing

The method of bacterial mediation of RNAi offers the possibility ofselectively targeting more than one gene at a time which might allow forincreased efficiency for future applications, e.g. anticancer treatmentthrough interference with multiple oncogenic pathways. To test thefeasibility of such an approach, both the mutated k-Ras oncogene andβ-catenin were targeted simultaneously. After simultaneous treatmentwith SL-siRAS and SL-siCAT, knockdown of both genes was observed at theprotein level and resulted in further decreased viability and colonyformation ability (FIG. 2). These findings demonstrate that the proposedconcept of bacterial mediated gene silencing can be successfully used invitro for different target genes and in different cell lines.

A mouse model was chosen to test whether this approach can be used tosilence target genes in vivo. CgTg5-Nagy mice express high levels of GFPin all tissues. 14 mice were randomly assigned to receive four doses of10⁶ cfu of either SL-siGFP or SL-siRAS i.v. on alternating days (sevenanimals per group). This treatment was well tolerated with no weightloss or clinically apparent signs of disease. All mice were sacrificedone day after the last treatment.

Liver tissue slides were assessed by fluorescence microscopy andimmunohistochemistry with GFP antibody. (FIG. 3A). Intravenous treatmentwith SL-siGFP led to decrease of fluorescence in the liver sections ofthe treated animals compared with SL-siRAS treated control animals.Histochemistry staining, with anti-GFP antibody, verified that changesin fluorescence were caused by a reduction in GFP and not caused bychanges in background fluorescence levels. (FIG. 5) To verify thatreductions in fluorescence in the liver sections of treated mice arereally caused by changes in GFP expression levels and not due to changesin background fluorescence, tissue slides were stained with GFP specificantibody.

Immunhistochemical staining patterns correlate well with fluorescencemicroscopy images and confirm that changes in fluorescence are caused bydecreased GFP expression. Fluorescence microscopy (50×) andcorresponding immunhistochemistry image (50×) of liver section fromcontrol (top row) and iv treated (lower row) animal.

Staining patterns correlated well with fluorescence images. Subsequentimage analysis revealed reductions in the number of GFP expressing cellsbetween 9-25% after SL-siGFP treatment. These findings were confirmed byflow cytometric analysis of single cell suspensions of hepatocytes whichshowed a significant decrease in the number of GFP-positive hepatocytesin SL-siGFP treated vs SL-siRAS treated animals (FIG. 3B). Flowcytometry measurements of hepatocyte and splenocyte suspensions wereperformed. After intravenous treatment with SL-siGFP, the number of GFP+hepatocytes was significantly reduced compared to control treated(SL-siRAS) animals (SL-siRAS: 50.0% [45.4-53.2%], SL-siGFP: 39.9%[26.1-53.2%], p<0.05).

These results indicate that significant gene silencing can be achievedin vivo using this approach. Using iv application of attenuated S.typhimurium we were able to extend the in vitro findings into a mousemodel and achieve significant gene silencing in the liver. Other organsmight become accessible through use of different invasive bacterialstrains or different routes of application. Especially professionalphagocytes will be a promising target for bacteria-mediated genesilencing, as transfection efficiencies have been reported to be higherfor these cells compared to cells of epithelial lineage.

Example 4 Transkingdom RNA Interference

The use of bacteria-mediated RNAi in higher organisms holds thepotential for functional genomics in mammalian system, as previouslydemonstrated in C. elegans, and for other in vivo applications of RNAi.To investigate this possibility, the bacterial plasmid pT7RNAi-Hly-Inv,termed TRIP (transkingdom RNA interference plasmid) was constructed(FIG. 6A). In this novel plasmid construct, the expression of shRNA wasdirected under the bacteriophage T7 promoter (Milligan and Uhlenbeck,Methods Enzymol. 180, 51-62 (1989) and Milligan et al., Nucleic AcidsRes. 15, 8783-8798 (1987), rather than a mammalian promoter or enhancer.The shRNA can only be produced by the bacterial system. The TRIP vectorcontains the Inv locus that encodes invasion (Isberg et al., Cell 50,769-778 (1987)), which permits the non-invasive E. coli to enterβ1-integrin-positive mammalian cells (Young et al., J. Cell Biol. 116,197-207 (1992)). The TRIP vector also contains the Hly A gene thatencodes listeriolysin O to permit genetic materials to escape from entryvesicles (Mathew et al., Gene Ther. 10, 1105-1115 (2003) andGrillot-Courvalin et al., Nat. Biotechnol. 16, 862-866 (1998)). TRIPconstructs were introduced into a competent strain of non-pathogenic E.coli, BL21DE3, which contains T7 RNA polymerase to express shRNA. A TRIPagainst the cancer gene β-catenin was constructed as an example.Activation of the β-catenin pathway from over-expression or oncogenicmutation of β-catenin is responsible for the initiation of the vastmajority of colon cancers and is involved in a variety of other cancertypes (Kim et al., Oncogene 24, 597-604 (2005)). Despite the potentialof β-catenin as a cancer therapeutic target, the β-catenin pathway hasbeen recalcitrant to inhibition by small molecules. β-catenin is apreferred choice in proof of concept experiments for testing the potencyof new a RNAi approach because it is commonly stabilized in cancercells. TRIP can be modified to enable bacteria to express interferingRNA against various genes of interest.

To determine if gene silencing can be achieved through this transkingdomsystem, human colon cancer cells (SW 480) were co-cultured in vitro withE. coli for 2 h (FIGS. 6B and 6D) or different time (FIG. 6C), thentreated with antibiotics to remove extracellular bacteria. Cells werefurther cultured for 48 h before harvest for analysis of gene silencing.As shown in FIG. 6B-6D, β-catenin was potently and specifically silencedat protein and mRNA level, while β-actin, k-Ras, andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) were not affected. Tofurther test the specificity of the transkingdom RNAi, E. colicontaining the TRIP against mutant k-Ras (GGT→GTT at codon 12) silencedk-Ras expression in SW 480 cells with the same codon 12 mutation, butnot in DLD1 cells with mutation in a different codon of k-Ras (GGC→GACat codon 13, FIG. 6E). As an shRNA control, E. coli containing the TRIPagainst wild type k-Ras exerted no gene-silencing effect on mutatedk-Ras in SW 480 cells (FIG. 6F). These results show that thetranskingdom RNA interference is highly gene-specific, sufficient todiscriminate a point mutation.

To investigate the variables that affect the potency of gene silencingby the transkingdom system, cells were incubated for 2 h with the E.coli at different multiplicity of infection (MOD. As shown in FIG. 6B,the potency of gene silencing was dependent on MOI, with near completegene silencing at a MOI of 1:1,000. To determine the effect ofco-culture time on gene silencing, cells were incubated with the E. coliat a MOI of 1:500 for different times. As shown in FIG. 6C,gene-silencing potency increased with incubation time up to 2 h. Thedependency of gene silencing on MOI and co-culture time providescontrollable flexibility for gene silencing in various applications.

To further confirm that the β-catenin gene silencing is mediatedspecifically by shRNA, identification of the specific cleavage fragmentof β-catenin mRNA was attempted by using 5′-RACE (rapid amplification ofcDNA ends) PCR technique. A specific hallmark of RNAi-mediated genesilencing is the cleavage of β-catenin mRNA at the specific sites of themRNA as predicted from the shRNA sequence. Based on the time course ofβ-catenin silencing (FIG. 7A), total RNA was isolated from SW 480 cells8 h and 16 h after treatment with E. coli expressing shRNA againstβ-catenin to identify the cleaved fragments of mRNA. The cleavedβ-catenin mRNA was found as early as 8 h after treatment with E. coliexpressing shRNA: no fragments were detected in the control (FIGS. 7Band 7C). The sequence analysis of the cleaved intermediate of β-cateninmRNA confirms the cleavage site located within the targeting sequence.This result shows that shRNA produced by bacteria trigger specificcleavage of the β-catenin mRNA through the RNAi-mediated gene silencing.

Induction of interferon response has been reported as a potentialchallenge to the specificity of some RNAi approaches (Bridge et al.,Nat. Genet. 34, 263-264 (2003) and Hornung et al., Nat. Med. 11, 263-270(2005)). To test if the gene silencing induced by the transkingdom RNAiis associated with interferon response induction, key interferonresponse genes were measured. The 2′,5′-oligoadenlylate synthetases(OAS1 and OAS2) are important interferon-induced genes for theinhibition of cellular protein synthesis after viral infection. MX1gene, a member of the interferon-induced myxovirus resistance proteinfamily (MX proteins), participates in the innate host defense againstRNA viruses. IFITM1, a member of the interferon-inducible transmembraneproteins, mediates the anti-proliferation activity of interferon. ISGF3γis part of a cellular interferon receptor involved in interferon-inducedtranscription regulation and stimulation. These genes have been used asa standard panel for analyzing interferon response induction byinterfering RNA (Interferon Response Detection Kit, SBI SystemsBiosciences, CA). The mRNA of the five-interferon response genes wereanalyzed with semiquantitative RT-PCR. As shown in FIG. 7D, no inductionof OAS1, OAS2, MX1, ISGF3γ and IFITM1 was detected following treatmentwith E. coli encoding shRNA against β-catenin. These data show that genesilencing induced by transkingdom RNAi is not associated withnon-specific interferon response induction.

The mechanism of the transkingdom RNAi transfer was investigated. Todetermine if cellular entry of E. coli is required to induce RNAi, thegene-silencing activity of E. coli was compared with or without the Invlocus. The Inv encodes invasin that interacts with β1-integrin tofacilitate the entry of E. coli into the cells. As expected, E. coliwithout Inv failed to enter cells (FIG. 8A). Surprisingly, Inv alone isnot sufficient to enable E. coli to enter colon cancer cells (FIG. 8A),and no detectable gene silencing was observed in the absence ofintracellular bacteria (FIG. 8B). The Hly A gene was introduced, whichis thought to facilitate delivered genetic materials to escape from theentry vesicles (Grillot-Courvalin et al., Nat. Biotechnol. 16, 862-866(1998). As expected, Hly alone failed to enable cell entrance of E.coli, but commensal E. coli with both Inv and Hly entered colon cancercells with high efficiency (FIG. 8A). Under these conditions β-cateninwas potently silenced up to 96 h (FIG. 8C). These results show that E.coli require both Inv and Hly to enter cells to induce transkingdomRNAi.

To determine whether gene silencing requires continued bacterialreplication inside target cells, tetracycline was employed to blockintracellular bacterial replication and gentamycin to removeextracellular bacteria. SW 480 cells were incubated with E. coli for 2 hfollowed by tetracycline treatment initiated at different times. Asshown in FIG. 8D, following the initial 2 h infection time, anadditional 2 h incubation time without tetracycline induced near maximumgene silencing; further delay in tetracycline treatment had no furtherenhancing effect on the degree of gene silencing. Surprisingly, therewas no evidence of significant intracellular bacterial replication inthe absence of tetracycline at 6 h and 48 h (FIG. 8E), which is likelydue to the function of lysosomes and other intracellular anti-bacterialmechanisms (Roy et al., Science 304, 1515-1518 (2004) and Battistoni etal., Infect. Immun. 68, 30-37 (2000). These results show thattranskingdom RNAi is not dependent on persistent bacterial replicationinside target cells after the initial infection (2 h) and incubationtime (2 h).

It was next determined if the transkingdom RNAi approach works in vivo.E. coli expressing shRNA against β-catenin were administered to miceorally. An inoculum of 5×10¹⁰ was administered orally five times perweek, which is comparable to a human dosage of the probiotic E. coliNissle 1917. Most of the inoculum is eliminated during passage throughthe bactericidal environment in the upper GI tract. Mice were treatedwith E. coli expressing shRNA against mouse β-catenin or with E. colicontaining the corresponding plasmid vector. Treatment was continued forfour weeks before the analysis of gene silencing byimmunohistochemistry. As shown in FIGS. 9A and 9B, β-catenin expressionwas silenced in the intestinal epithelium by E. coli expressingβ-catenin shRNA (P<0.01), not by the control E. coli. As a control,GAPDH expression was not reduced (FIG. 10). The gene silencing effectwas more pronounced in the regions of or adjacent to the Peyer's patches(FIG. 9B). Treatment was well tolerated with no gross or microscopicsigns of epithelial damage or ulcerations (FIG. 9B). These results showthat mammals respond to E. coli expressing specific shRNA with powerfullocal RNAi in vivo.

The transkingdom RNAi approach was investigated to determine if it canbe used to silence a disease gene after systemic dosing. Intravenousadministration of therapeutic bacteria has been tested in clinicaltrials with demonstrated safety in cancer patients (Toso et al., J.Clin. Oncol. 20, 142-52 (2002)). Nude mice with xenografted human coloncancer were treated intravenously with of 10⁸ cfu of E. coli encodingshRNA against human β-catenin. Three doses were given at a 5-dayinterval. The treatments were well tolerated without adverse effects. Asshown in FIG. 9, treatment with E. coli encoding shRNA against β-cateninresulted in significant decrease in β-catenin mRNA (p<0.005, FIG. 9C)and protein (p<0.01, FIGS. 9D and 9E) in the tumor tissues. These datashow that bacteria-mediated transkingdom RNAi can silence a disease genein a distant part of the body after systemic administration.

These results show that gene silencing can be achieved through atranskingdom system. Importantly, the potency and specificity of RNAi ispreserved. Non-pathogenic E. coli has been used clinically as probioticswith demonstrated safety (Rembacken et al., Lancet 354, 635 (1999)).Therefore, this transkingdom system provides a practical and clinicallycompatible way to deliver RNA interference for medical indications. ThisE. coli-based RNAi technology also provides a convenient vector systemfor conducting RNAi-based functional studies of genes. Finally, theresults invite an intriguing possibility that such exchange ofinterfering RNA may occur in nature under cohabitive, infectious, orsymbiotic conditions.

1. A method for delivering one or more siRNAs to animal cells, themethod comprising infecting the animal cells with live invasive bacteriacontaining one or more siRNAs or one or more DNA molecules encoding oneor more siRNAs.
 2. A method for regulating gene expression in animalcells, the method comprising infecting the animal cells with liveinvasive bacteria containing one or more siRNAs or one or more DNAmolecules encoding one or more siRNAs, wherein the expressed siRNAsinterfere with the mRNA of the gene to be regulated, thereby regulatingexpression of said gene.
 3. A method for treating or preventing canceror a cell proliferation disorder in a mammal, the method comprisingregulating the expression of a gene in a cell known to increase cellproliferation by infecting the cells of the mammal with live invasivebacteria containing one or more siRNAs or one or more DNA moleculesencoding one or more siRNAs, wherein the one or more siRNAs are targetedto reduce expression of the gene.
 4. (canceled)
 5. (canceled)
 6. Themethod of claim 1, wherein said live invasive bacteria arenon-pathogenic or non-virulent bacteria.
 7. (canceled)
 8. The method ofclaim 6, wherein said live invasive bacteria are an attenuated strainselected from a member of the group consisting of Listeria, Shigella,Salmonella, E. coli, and Bifidobacteriae.
 9. (canceled)
 10. (canceled)11. (canceled)
 12. The method of claim 1, wherein said live invasivebacterium is a member of the group consisting of Yersinia spp.,Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp.,Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacterspp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacteriumspp., Legionella spp., Rhodococcus spp., Pseudomonas Spp., Helicobacterspp., Salmonella spp., Vibrio spp., Bacillus spp., Leishmania spp. andErysipelothrix spp. which have been genetically engineered to mimic theinvasion properties of Shigella spp., Listeria spp., Rickettsia spp., orenteroinvasive E. coli spp.
 13. (canceled)
 14. The method of claim 1,wherein said animal cell is a mammalian cell.
 15. (canceled)
 16. Themethod of claim 14, wherein said mammalian cell is a human cell.
 17. Themethod of claim 3, wherein said mammal is a human.
 18. The method ofclaim 1, wherein said one or more DNA molecules encoding said one ormore siRNAs are transcribed within the animal cell.
 19. The method ofclaim 18, wherein said one or more siRNAs are transcribed within theanimal cell as shRNAs.
 20. The method of claim 18, wherein said one ormore DNA molecules encoding said one or more siRNAs comprise anRNA-polymerase III promoter.
 21. The method of claim 20, wherein saidRNA-polymerase III promoter is a U6 promoter or an H1 promoter.
 22. Themethod of claim 1, wherein said one or more DNA molecules encoding saidone or more siRNAs are transcribed within the bacterium.
 23. The methodof claim 22, wherein said one or more DNA molecules encoding one or moresiRNAs comprise a prokaryotic promoter.
 24. The method of claim 23,wherein said prokaryotic promoter is a T7 promoter.
 25. The method ofclaim 1, wherein said one or more DNA molecules are introduced to thecell through type III export or bacterial lysis.
 26. The method of claim25, wherein said bacterial lysis is triggered by the addition of anintracellular active antibiotic.
 27. The method of claim 26, whereinsaid antibiotic is tetracycline.
 28. The method of claim 25, whereinsaid bacterial lysis is triggered through bacterial metabolicattenuation.
 29. The method of claim 28, wherein said metabolicattenuation is auxotrophy.
 30. (canceled)
 31. (canceled)
 32. (canceled)33. (canceled)
 34. The method of claim 3, wherein expressed siRNAsinterfere with the mRNA of the gene to be regulated.
 35. The method ofclaim 2, wherein the expressed siRNAs direct the multienzyme complexRNA-induced silencing complex of the cell to interact with the mRNA ofthe gene to be regulated.
 36. (canceled)
 37. The method of claim 35,wherein expression of the gene is decreased or inhibited.
 38. The methodof claim 2, wherein said gene is ras or β-catenin.
 39. The method ofclaim 38, wherein said ras is k-Ras.
 40. The method of claim 3, whereinsaid cell is a colon cancer cell or a pancreatic cancer cell. 41.(canceled)
 42. (canceled) 43-52. (canceled)